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European Journal of Pharmaceutical Sciences 77 (2015) 122–134
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European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier .com/ locate /ejps
Development, optimization and biological evaluation of chitosan scaffoldformulations of new xanthine derivatives for treatment of type-2diabetes mellitus
http://dx.doi.org/10.1016/j.ejps.2015.06.0080928-0987/� 2015 Elsevier B.V. All rights reserved.
⇑ Corresponding author.E-mail addresses: [email protected] (F.G. Lupascu), mamoni.dash@
ugent.be (M. Dash), [email protected] (S.K. Samal), [email protected] (P. Dubruel), [email protected] (C.E. Lupusoru), [email protected] (R.-V. Lupusoru), [email protected] (O. Dragostin), [email protected] (L. Profire).
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Florentina Geanina Lupascu a, Mamoni Dash b, Sangram Keshari Samal c, Peter Dubruel b,Catalina Elena Lupusoru d, Raoul-Vasile Lupusoru d, Oana Dragostin a, Lenuta Profire a,⇑a University of Medicine and Pharmacy ‘‘Grigore T. Popa’’, Faculty of Pharmacy, University 16, 700115 Iasi, Romaniab Polymer Chemistry & Biomaterials Research Group, Ghent University, Krijgslaan 281, S4-Bis, B-9000 Ghent, Belgiumc Laboratory of General Biochemistry and Physical Pharmacy, Centre for Nano- and Biophotonics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgiumd University of Medicine and Pharmacy ‘‘Grigore T. Popa’’, Faculty of Medicine, University 16, 700115 Iasi, Romania
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Article history:Received 16 April 2015Received in revised form 4 June 2015Accepted 12 June 2015Available online 12 June 2015
Keywords:Diabetes mellitusChitosanMicroparticlesXanthine derivativeDrug delivery
New xanthine derivatives as antidiabetic agents were synthesized and new chitosan formulations havebeen developed in order to improve their biological and pharmacokinetic profile. Their physicochemicalproperties in terms of particle size, morphology, swelling degree, crystalline state, the loading efficiencyas well as in vitro release and biodegradation rate were evaluated. According to the results the opti-mized formulations have a high drug loading efficiency (more than 70%), small particle size, a goodrelease profile in the simulated biological fluids (the percentage of cumulative release being more than55%) and improved biodegradation rate in reference with chitosan microparticles. The presence ofxanthine derivatives (6, 7) in chitosan microparticles was demonstrated by means of FTIR analysis.The X-ray diffraction (XRD) proved that xanthine derivatives present a crystalline state. The biologicalevaluation assays confirmed the antioxidant and antidiabetic effects of the xanthine derivatives (6, 7)and their chitosan formulations (CS-6, CS-7). Xanthine derivative 6 showed a high antiradical scaveng-ing effect (DPPH remaining = 41.78%). It also reduced the glucose blood level with 59.30% and recordedlevel of glycosylated hemoglobin was 4.53%. The effect of its chitosan formulation (CS-6) on the level ofblood glucose (114.5 mg/dl) was even more intense than the one recorded by pioglitazone (148.5 mg/dl)when used as standard antidiabetic drug. These results demonstrated the potential application of xan-thine derivative 6 and its chitosan formulation (CS-6) in the treatment of the diabetes mellitussyndrome.
� 2015 Elsevier B.V. All rights reserved.r Pers
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1. IntroductionDiabetes mellitus type 2 (T2DM) is the most common form ofdiabetes, which is strongly associated with a sedentary life styleand obesity (Dandona and Aljada, 2004). T2DM is a complex meta-bolic syndrome affecting more than 100 millions of people from allover the world, being considered one of the five causes of worldmortalities (Shanmugam et al., 2011). There are two factors that
are strongly related to hyperglycemia: insulin resistance in theliver, the adipose tissue and the skeletal muscles and insulin defi-ciency caused by the pancreatic b-cell dysfunction (Jin et al., 2008).Current antidiabetic therapy include four classes of drugs: sulpho-nyureas and glinides, biguanides, thiazolidinediones anda-glucosidase inhibitors. Unfortunately, all current drugs, are fre-quently associated with several side effects as weight gain, edema,anemia, heart failure, fracture of bones and gastrointestinal intol-erance (Cariou et al., 2012; Quin et al., 2008).
The newly discovered incretin effect has provided a new path oftreatment which is able to reduce the hyperglycemia with minimalside effects. The incretin hormones called glucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic peptide (GIP)increase glucose-dependent stimulation of insulin secretion,suppress abnormally elevated glucagon secretion from pancreatic
F.G. Lupascu et al. / European Journal of Pharmaceutical Sciences 77 (2015) 122–134 123
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a-cell, delay gastric emptying, and promote satiety, weight loss, aswell as b-cell growth and inhibit b-cell apoptosis in animal models(Tsai et al., 2009). Unfortunately, after secretion, GIP and GLP-1 arevery fast inactivated by dipeptidyl peptidase 4 (DPP-4), theirhalf-lifes being 5 min (GIP) and 2 min (GLP-1) respectively(Arulmozhi and Portha, 2006). The latest therapeutic approacheson incretins action include GLP-1 receptor agonists with increasedresistance to DPP-4 degradation and inhibitors of DPP-4(Arulmozhi and Portha, 2006; Akarte et al., 2012).
On the other hand the xanthine derivatives are compoundswith purine core and which represents a very important struc-tural unit in drug discovery. A survey of literature reveals the bio-logical properties of this structure including hypoglycemic,anticancer, antioxidant, anti-inflammatory, bronchodilator andxanthine oxidase inhibitory effects (Beedkar et al., 2012). Theresearches focused on DPP-4 inhibitors leaded to discover ahighly potent, selective and long acting inhibitor (linagliptin) thatreceived approval in 2011. Used as monotherapy (5 mg/day) orcombined with other agents, linagliptin showed a significantreduction of glycated hemoglobin (HbA1c) and a jèun glucoselevel (Gallwitz 2012; Hoimark et al., 2012). This compoundshowed also the xanthine oxidase inhibition activity in vitro andthe serum uric acid reducing in type 2 diabetic patients in vivo(Jones et al., 2014; Yamagishi et al., 2014). At the same timexanthine derivatives are known to have antioxidant and radicalscavenging activity (Bhat and Madyastha, 2001). These effectsare very important because it is known the implication of reactiveoxygen species in many diseases including diabetes, myocardialinfarction, neurological disorders, asthma, cancer, rheumatoidarthritis (Jin et al., 2008; Kangralkar et al., 2010). Particularly indiabetes the high level of blood glucose is the main cause relatedto increased production of free radicals, especially reactive oxy-gen species resulted from glucose auto-oxidation, lipid peroxida-tion and protein glycosylation. Endothelial cells are chronicallyexposed to oxidative stress leading to the initiation and progres-sion of microvascular and macrovascular complications of dia-betes (Lupascu et al., 2013; Pasupathi et al., 2009). Related toantitumor effect, xanthine derivatives proved favorable effect forvarious carcinogens in numerous organs including skin, lung,stomach and liver (Motegi et al., 2013). Isbufylline, a xanthinederivative, showed also a significant anti-inflammatory and bron-chodilator action through inhibition of platelet activating factor(PAF) (Manzini et al., 1993).
The aim of this study was to design new xanthine derivativesand to improve their pharmacokinetic profile using chitosan basedpolymer matrix as a new potential strategy in diabetes mellitustherapy.
Polymer matrices based on chitosan, polyisobutylene, polyiso-prene, Carbopol 934P, etc. (Guo, 1994; Lowe et al., 1999) are usu-ally used to improve the bioavailability of the drugs and also toobtain a sustained release of the drug (Hansen et al., 1992; Yuand Grainger, 1995). The desirable properties of an oral deliverysystem for prolonged release are high drug loading capacity, goodmucoadhesion and high tolerance (Knuth et al., 1993; Senel et al.,2000).
Chitosan is a linear polysaccharide, o polymer of a(1 ? 4)-linked 2-amino-2-deoxy--D-glucopyranose, being acopolymer of N-acetylglucosamine and glucosamine. Its propertiessuch as hydrophilicity, non-toxicity, biodegradability, biocompati-bility and bioadhesive support its use as an important pharmaceu-tical excipient (Rao and Sharma, 1997; Dash et al., 2011). Chitosanhas also important biological effects such as hypobilirubinaemicand hypocholesterolemic effects, antacid and antiulcer activities(Sinha et al., 2004). Moreover, recent studies have shown theanti-inflammatory, antioxidant, antidiabetic and neuroprotective
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effects of chitosan (Jean et al., 2012; Lee et al., 2003; Ju et al.,2010; Kim et al., 2009).
2. Materials and methods
2.1. Materials
Chitosan and TPP were purchased from Sigma Aldrich(Belgium). The characteristics of chitosan were as follow: molecu-lar weight (Mw 190,000–375,000), viscosity (200 cps) and degree ofdeacetylation (DD 75–85%). Lysozyme (hen egg-white) was alsopurchased from Sigma–Aldrich (Belgium). Theophylline anhydrous99%, ethyl chloroacetate 99%, hydrazine hydrate 98%,4-bromophenyl isothiocyanate 98%, chloroacetyl chloride 99%,4-chlorobenzaldehyde 97%, 4-hydroxybenzaldehyde 97% and2,2-diphenyl-1-picrylhydrazyl 95% were purchased from Sigma–Aldrich (Romania). All other reagents and solvents had puritygrade and were obtained from commercial suppliers. MaleWistar rats and Swiss mice were purchased from the Biobase of‘‘Grigore T. Popa’’ University of Medicine and Pharmacy, Iasi,Romania.
2.2. Synthesis
The 1H NMR and 13C NMR spectra were recorded in DMSO-d6
with a Bruker Avance 300 MHz instrument. The chemical shiftswere expressed in ppm using tetramethylsilane (TMS) as internalstandard. FT-IR spectra were performed on a Biorad FT-IR- FTS575C spectrometer. All melting points were determined on BuchiMelting Point B-540 apparatus.
2.2.1. Synthesis of xanthine-thiazolidine derivatives (6, 7)2.2.1.1. Synthesis of (1,3-dimethylxanthin-7-yl)ethyl acetate(2). 9.76 g (79 mmol, 8.5 ml) of ethyl-chloroacetate was added toa solution of 1,3-dimethyl-xanthine sodium salt (1) (16.1 g,79 mmol) in mixture of ethanol (100 ml) and dimethylformamide(DMFA) (50 ml). The reaction mixture was heated under refluxfor 7 h. After cooling the solid product was filtered, dried andrecrystallized from ethanol. The reaction was monitored by TLCon silica gel using dichloromethane: methanol (25:1) system. Thecompound was obtained by adapting similar methods describedin the speciality literature (Kus� et al., 2008; Amr et al., 2009;Bhati and Kumar, 2008). White powder; yield 96%; mp:140–143 �C; FT-IR (KBr, cm�1): 1755 (AC@O ester), 1659(AC@N), 1469 (ANACH2), 1432 (ACH2Aester), 1369 (ACH3 ester),1294 (CAOAC), 1217 (ACAN); 1H NMR d/ppm (400 MHz, DMSO):8.03 (s, 1H, ACH@N); 5.17 (s, 2H, NACH2A); 4.17 (q, 2H,ACH2A); 3.44, 3.20 (s, 6H, 2CH3); 1.21 (t, 3H, ACH3); 13C NMRd/ppm (101 MHz, DMSO): 167.74 (C9), 154.71 (C6), 151.58 (C2),148.16 (C4), 143.51 (C7), 106.64 (C5), 61.69 (C10), 47.41 (C8),29.75 (C3), 27.58 (C1), 14.56 (C11).
2.2.1.2. Synthesis of (1,3-dimethylxanthin-7-yl)acethyl hydrazine(3). 11.56 g (228 mmol, 11.43 ml) of hydrazine hydrate was addedto a solution of 1,3-dimethyl-xanthine-7-yl-ethyl acetate (2)(20.5 g, 76 mmol) in ethanol (300 ml), and then the mixture wasrefluxed for 5 h. Afterwards the mixture of reaction was cooledand filtered to obtain a white powder. The crude product wasrecrystallized from ethanol. The reaction was monitored by TLCon silica gel using dichloromethane:methanol (6:1) system. Thecompound was obtained by adapting similar methods describedin the speciality literature (Kus� et al., 2008; Amr et al., 2009;Bhati and Kumar, 2008). White powder; yield 89%; mp: 280–283 �C; FT-IR (KB, cm�1): 3438, 3307, 3299 (ANHANH2); 1661
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(AC@N), 1608 (AC@O amide), 1470, 1457 (ANACH2); 1230(ACAN); 1H NMR d/ppm. (300 MHz, DMSO): 9.34 (NH); 8.02 (s,1H, ACH@N); 4.95 (s, 2H, ACH2A); 4.28 (d, NH2); 3.45, 3.21 (s,6H, 2CH3); 13C NMR d/ppm (101 MHz, DMSO): 167.73 (C9),154.72 (C6), 151.58 (C2), 148.17 (C4), 143.50 (C7), 106.64 (C5),47.41 (C8), 29.75 (C3), 27.58 (C1).
2.2.1.3. Synthesis of 1-[2-(1,3-dimethylxanthin-7-yl)acetyl]-4-(4-bro-mophenyl)thiosemicar-bazide (4). The (1,3-dimethylxanthin-7-yl)acethyl hydrazine (3) (7 g, 23 mmol) was partially dissolved in mix-ture of dioxan (250 ml) and DMFA (100 ml), and then4-bromphenyl isothiocyanate (5.18 g, 23 mmol) was added. Thereaction mixture was heated under reflux, and after 10 min anabundant precipitate was obtained. After 6 h of reflux the precipi-tate was filtered and recrystallized from a mixture of ethanol anddioxane (3:1). The reaction was monitored by TLC using ethylacetate:acetone (2:1) system. The compound was synthesized byadapting similar methods described in the speciality literature(Kus� et al., 2008; Amr et al., 2009; Bhati and Kumar, 2008;Bondock et al., 2007). White powder; yield 60%; mp: 210–212 �C;FT-IR (KBr, cm�1): 3245 (ANHA), 1654 (AC@N), 1225 (ACAN),1531 (ANHAC@S), 1485 (ANACH2), 1130 (AC@S), 544 (CABr);1H NMR d/ppm (400 MHz, DMSO): 8.25 (s, 1H, ACH@N); 7.51,7.33 (d, 4H, ArAH); 5.77 (s, 2H, ACH2A); 3.45, 3.22 (s, 6H, 2CH3);13C NMR d/ppm (101 MHz, DMSO): 181.52 (C10), 164.52 (C9),154.92 (C6), 151.43 (C2), 150.56 (C4), 145.59 (C7), 136.19 (C11),132.09 (C13, C15), 128.76 (C12, C16), 119.15 (C14), 107.42 (C5),30.54 (C8), 32.39 (C3), 29.51 (C1).
2.2.1.4. Synthesis of 2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydra-zono}-3-(4-bromophe-nyl)thiazolidine-4-one (5). Chloroacetyl chlo-ride (3.1 ml, 39 mmol) was added slowly, at room temperature(exothermic reaction), to a mixture of 1-[2-(1,3-dimethylxanthin-7-yl)-acetyl]-4-(4-bromophenyl)thiosemicarbazide (4) (6.4 g,13 mmol) in 250 ml mixture of methanol – chloroform (1:1).After 10 h of heating under reflux a yellow clear solution wasobtained. The solvent was removed under reduced pressure andthe residue was precipitate in cold water. The solid was recrystal-lized from ethanol. The reaction was monitored by TLC usingdichloromethane:methanol (15:1) system. The synthesis was per-formed by adapting similar methods described in the speciality lit-erature (Bondock et al., 2007; Gouda and Abu-Hashem 2011; Cacicet al., 2006; Kalia et al., 2007). White-yellow powder; yield 52%;mp: 240–242 �C; FT-IR (KBr, cm�1): 1657 (AC@N), 1196 (ACAN),1596 (AC@O amide), 1451, 1407 (ANACH2), 3448 (ANH), 1724(AC@O thiazolidine-4-one), 810 (CASAC) cm�1; 1H NMR d/ppm(400 MHz, DMSO): 8.28 (s, 1H, ACH@N); 7.54, 7.48 (d, 4H,ArAH); 5.80 (s, 2H, ACH2AN); 5.08 (s, 2H, ACH2A thiazolidine);3.44, 3.23 (s, 6H, 2CH3); 13C NMR d/ppm (101 MHz,DMSO):173.12 (C9), 172.54 (C12), 154.91 (C6), 151.43 (C2), 150.48(C4), 148.42 (C10), 145.57 (C7), 134.45 (C13), 131.09 (C15, C17),123.84 (C15, C18), 118.73 (C16), 107.42 (C5), 30.28 (C8), 32.31 (C3),30.06 (C11), 29.43 (C1).
2.2.1.5. Synthesis of 2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydra-zono}-3-(4-bromo-phenyl)-5-(4-R-benzyliden)thiazolidine-4-one (6,7). The aromatic aldehyde (79 mmol) was added to a solution of2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-bromophenyl)thiazolidine-4-one (6) (4 g, 79 mmol) in dioxane(100 ml), using three drops of piperidine and two drops of glacialacetic acid as catalysts. The mixture of reaction was heating underreflux for 6 h and then the solvent was removed under reducedpressure. The solid was precipitated in cold water, filtered off,washed with cold water and recrystallized from 1,4 dioxane. Thereaction was monitored by TLC on silica gel using toluene:ethylacetate (3:1) system. The synthesis was performed by adapting
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similar methods described in the speciality literature (Bondocket al., 2007; Gouda and Abu-Hashem 2011; El-Gaby et al., 2009).
2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-bromophenyl)-5-(4-hydroxyben-zyliden)thiazolidine-4-one (6). Yellowpowder; yield 79%; mp: 265–267 �C; FT-IR (KBr, cm�1): 1643(AC@N), 1600 (C@C), 1227 (ACAN), 1595 (AC@O amide), 1467,1425 (ANACH2), 3248 (ANHA), 1700 (AC@O thiazolidine-4-one),775 (CH), 761 (CASAC), 498 (ACABr), 3112 (AOH); 1H NMRd/ppm (400 MHz, DMSO): 8.28 (s, 1H, ACH@NA); 7.57, 7.51 (d,4H, Ar-H); 7.54, 7.48 (d, 4H, ArAH); 7.14 (s, 1H, @CHA); 5.81 (s,2H, ACH2A); 3.44, 3.23 (s, 6H, 2CH3); 13C NMR d/ppm. (101 MHz,DMSO): 172.82 (C9), 167.25 (C12); 157.24 (C16), 154.44, 154.74(C2, C6); 150.95 (C4), 148.42 (C7), 139.63 (C13), 132.21, 132.45(C22, C24), 131.76 (C20), 127.85 (C14, C18, C19), 123.54, 123.82 (C21,
C25), 116.97, 117.12 (C15, C17), 119.29 (C23), 113.22 (C11), 107.74(C5), 30.06 (C8), 29.47, 27.5 (C1, C3).
2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-bromophenyl)-5-(4-chloroben-zyliden)thiazolidine-4-one (7). Brownpowder; yield 87%; mp: 272–273 �C; FT-IR (KBr, cm�1): 1643(AC@N), 1602 (C@C), 1595 (AC@O amide), 1468, 1426(ANACH2), 3248 (ANHA), 1700 (AC@O thiazolidine-4-one), 1229(ACAN), 761 (CASAC), 497.54 (ACABr), 6856 (ACACl) cm�1; 1HNMR d/p.p.m. (400 MHz, DMSO): 8.28 (s, 1H, ACH@NA); 7.57,7.52 (s, 4H, ArAH); 7.54, 7.48 (d, 4H, ArAH); 7.21 (s, 1H,@CHA);5.78 (s, 2H, ACH2A); 3.44, 3.23 (s, 6H, 2CH3); 13C NMR d/ppm(101 MHz, DMSO): 172.81 (C9), 165.27 (C12), 154.44, 154.74 (C2,
C6), 150.95 (C4), 148.42 (C7), 139.63 (C13), 133.43 (C16), 132.62(C22, C24), 131.75 (C20), 129.38, 129.64 (C15, C17), 127.47, 127.65(C14, C18), 125.53, 125.82 (C21, C25), 119.29 (C23), 113.22 (C11),105.85 (C5), 30.65 (C8), 29.47, 27.5 (C1, C3).Use
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2.3. Preparation of chitosan microparticles
Chitosan microparticles were prepared by the ionic gelation ofthe polycationic chitosan solution with anionic tripolyphosphate(TPP). Briefly, chitosan (low molecular weight – CSLMW and med-ium molecular weight – CSMMW) was dispersed in 100 ml ofacetic acid 2% (v/v) and the mixture was left under stirring over-night. Then, the chitosan solution was dropped through a syringeneedle (18 G) into 5 ml of TPP solution (pH 9.0) under gently stir-ring when chitosan beads were formed instantaneously. The mix-ture was stirred at room temperature for 24 h in order to achievean efficient reticulation. Different concentrations of chitosan (1%,2%, w/v) and TPP (5%, 10%, w/v) were used to obtain the best for-mulation parameters and stable microparticles. The chitosan beadswere separated from TPP solution, washed three times with distil-lated water and dried at room temperature.
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2.4. Preparation of chitosan microparticles loaded with xanthinederivatives
The xanthine derivatives (6, 7) were loaded into chitosanmicroparticles using ionic gelation method. Briefly, 1 ml ofxanthine derivatives solution (6, 7) in acetic acid was mixed with2 ml of chitosan solution under gently stirring. After 8 h theresulted mixture was dropped through a syringe needle (18 G) into5 ml of TPP solution (5%, pH 9.0). The procedure was similar to themethod described above for chitosan microparticles. In order toobtain high loading efficiency and stable microparticles, three con-centrations for each xanthine derivative have been used:2.5 mg/ml, 5 mg/ml and 7.5 mg/ml (xanthine derivative 6) and0.5 mg/ml, 2.5 mg/ml and 5 mg/ml (xanthine derivative 7) respec-tively. It was also used two types of chitosan (low and mediummolecular weight).
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2.5. Characterization of chitosan microparticles loaded with xanthinederivatives
2.5.1. Particle size measurements and morphologyThe size of the microparticles (in wet and dry state) was
measured using the Zeiss (Axiotech) optical microscope (5 timesmagnification). The morphology of the microparticles was studiedby scanning electron microscopy (SEM) using a Desktop SEM(Phenom, The Nederlands). To improve the performance of SEManalysis the samples were coated with a thin gold layer (20 nm)using an Emitech K5550X device (Phenom, The Nederlands), oper-ated at 20 mA for 2 min.
2.5.2. Swelling degree (SD)The dynamic swelling experiments were performed in distil-
lated water and simulated gastric fluid (SGF) with pH 1.6 at 37 �C(in a thermostated water bath). The microparticles weight wasmeasured as a function of time. At different times microparticleswere removed from medium (water and SGF respectively), driedquickly and carefully with filter paper and weighted (W1). At theend of the experiment the microparticles were once again driedand weighted (W2). The experiments were performed in triplicateand average values were calculated. The degree of swelling at dif-ferent times was calculated using the following formula:
SD ð%Þ ¼W1 �W2=W2 � 100 ð1Þ
where:
W1 – the weight of the swollen microparticles;W2 – the weight of the dried microparticles.
2.5.3. X-ray diffraction (XRD) studiesThe crystallinity of the xanthine derivatives and of the chitosan
microparticles loaded with xanthine derivatives was evaluated byXRD measurements, using ARL X’ TRA Powder Diffractometer(Thermo Scientific). The scanning was done in a range of 10–50�(2h) with a step size of 0.02�.
2.5.4. Fourier transform infrared (FT-IR) spectroscopyFT-IR spectra of chitosan, xanthine derivatives and chitosan
microparticles loaded with xanthine derivatives were recordedusing Biorad FT-IR spectrometer FTS 575C in the range between4000 cm�1 and 500 cm�1, after 32 scans at a resolution of4 cm�1. The spectra processing was carried out with the HorizonMB™ FT-IR Software.
2.5.5. Loading efficiency (LE)The loading efficiency of xanthine derivatives (6, 7) into chi-
tosan microparticles was evaluated using UV spectrophotometricmethod (UVIKNO XL, BIOTECH Instruments). The content of thexanthine derivatives in the TPP solution was evaluated spectropho-tometric at 280 nm, after removing the chitosan beads. A standardcurve for each xanthine derivatives with a correlation coefficient ofR2 = 0.999 was used. The loading efficiency (%) was calculatedusing the following formula:
% loading efficiency ¼ C1=C0 � 100 ð2Þ
where:
C0 = initial concentration of the xanthine derivatives (mg/ml);C1 = concentration of the xanthine derivatives in the TPP solu-tion (mg/ml).
2.5.6. In-vitro releaseIn vitro release of xanthine derivatives (6, 7) was studied using
three types of simulated biological fluids: simulated gastric
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fluid – SGF (pH 1.6), simulated intestinal fluid – SIF (pH 6.5) andsimulated colonic fluid – SCF (pH 6.5) (Martin et al., 2002). Aweighed amount of chitosan microparticles loaded with xanthinederivatives was placed in a flask containing 2 ml of release medium,according to the procedure described in the speciality literature(Gallardo et al., 2001). The sample was incubated at 37 ± 0.1 �Cunder constant stirring at 100 rpm. At different times, a sample of2 ml was collected from the release medium and replaced by an equalvolume of fresh medium The concentration of the xanthine deriva-tives in the solution was evaluated spectrophotometric at 280 nmusing a standard curve with correlation coefficient of R2 = 0.999. Therelease rate (%) was calculated according to the following formula:
% release rate ¼ A� B=A� 100 ð3Þ
where:
A = initial concentration of the xanthine derivatives (mg/ml) inthe release medium;B = concentration of the xanthine derivatives (mg/ml) in therelease medium at different times.
2.5.7. In-vitro biodegradationIn vitro degradation of chitosan microparticles loaded with xan-
thine derivatives was performed using phosphate buffered salinewith pH value of 7.4 (PBS, 1 ml) containing 1.5 lg/ml lysozymeat 37 �C. The concentration of lysozyme was selected to be similarto the concentration from the gastric fluid (Wang et al., 2009).Briefly, an amount of dried chitosan microparticles loaded withxanthine derivatives were incubated with lysozyme solution undergentle stirring (100 rpm). The lysozyme solution was refresheddaily to ensure continuous enzyme activity (Masuda et al., 2001).On the 3rd, 5th and 7th day the loaded chitosan microparticleswere separated from the medium, rinsed with distilled water andweighed. The in vitro degradation, expressed as percentage ofweight loss, was calculated according to the following formula(Dash et al., 2009):
% Degradation ¼ G0 � Gt=G0 � 100 ð4Þ
where:
G0 = the weight of the chitosan microparticles at the beginningof the experiment (the 1st day);Gt = the weight of the chitosan microparticles at different times(on the 3rd, 5th, 7th day).
2.6. Biological evaluation
2.6.1. In vitro assay2.6.1.1. DPPH radical scavenging assay. The radical scavenging activ-ity of the xanthine derivatives (6, 7) toward the radical1,1-diphenyl-2-picrylhydrazyl (DPPH) was evaluated as describedin the speciality literature (Osorio et al., 2012; Jeong et al., 2004)with slight modifications. The sample of compounds (1 ml,100 lM in DMSO) was mixed with 2 ml solution of DPPH(0.1 mM in methanol). The absorbance of the sample (As) was mea-sured at room temperature, in the dark, at 517 nm, every 5 min, fora period of 40 min. The methanol solution of DPPH was used ascontrol sample (Ac). All measurements were performed in tripli-cate. The radical scavenging capacity was calculated according tothe following formula:
% DPPH remaining ¼ 100� ðAs=AcÞ ð5Þ
where:
As = absorbance of the sample at different times;Ac = absorbance of the control sample.
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Fig. 1. Synthesis of xanthine-thiazolidine derivatives (6, 7).
126 F.G. Lupascu et al. / European Journal of Pharmaceutical Sciences 77 (2015) 122–134
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2.6.2. In vivo assays2.6.2.1. Acute toxicity assay. The acute toxicity of xanthine deriva-tives (6, 7) was evaluated with the lethal dose test which consistin administration of the compounds at increasing doses in orderto establish the dose that would kill 50% of the mice (LD50). Themice (25–30 g) were housed in polyethylene cages (no more thanfour animals per cage), at constant temperature (24 ± 2 �C) with12 h light and 12 h dark cycle and relative humidity of 40–70%.The animals were acclimatized to laboratory conditions for oneweek before the experiment and receiving standard food. The micewere kept fasting for 24 h before the experiment with water ad libi-tum. Each group (six mice/group) was treated orally (p.o.) withxanthine derivatives as emulsion in Tween 80 (50 mg/ml). Thesymptoms of toxicity and mortality rate were noticed at 24 h,48 h, 72 h, 7 days and 14 days after administration. The LD50 ofcompounds was estimated according to the following formula(Akhila et al., 2007):
LD50 ¼ LD100 �Rða� bÞ
nð6Þ
where
a = the difference between two successive doses of the testedcompound;b = the arithmetic average of the animals from two successiveseries that died;n = the number of animals per group;LD100 = the 100% lethal dose.
The experiment was carried out in accordance with the currentguidelines for the veterinary care of laboratory animals and wereperformed with the consent of the Ethics Committee for AnimalResearch of ‘‘Grigore T. Popa’’ University of Medicine andPharmacy Iasi (no 291/2013).
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2.6.2.2. Antidiabetic effects.2.6.2.2.1. Streptozotocin induced diabetes. Male Wistar rats weigh-ing 150–200 g were used for induced diabetes model. The animalswere housed at constant temperature (24 ± 2 �C) with 12 h lightand 12 h dark cycle and relative humidity of 40–70%. They receivedstandard food and water ad libitum. The rats were kept fasting for24 h before the experiment with water ad libitum. A solution ofstreptozotocin (30 mg/ml in 0.01 M citrate buffer, pH 4.5) wasintraperitoneally administered in a single dose of 65 mg/kg(Kumar et al., 2012). A control group which received only citratebuffer was used. On the 1st, 2nd, 4th and 7th day, the concentra-tion of blood glucose was measured using glucometer FORAG71a, Switzerland with FORA test strips. Only animals with hyper-glycemia (blood glucose level > 250 mg/dl) were used for furtherinvestigations.2.6.2.2.2. Experimental design. The animals were divided into 7groups (7 animals per group): group 1 received chitosan micropar-ticles loading with compound 6 (CS-6, 81 mg/kg); group 2 receivedchitosan microparticles loading with compound 7 (CS-7,80 mg/kg); group 3 received compound 6 (81 mg/kg); group 4received compound 7 (80 mg/kg); group 5 received pioglitazoneas standard drug (4 mg/kg); group 6 received vehicle (Tween 80–2 ml/kg); group 7 non-diabetic animals received vehicle (Tween80–2 ml/kg). The tested compounds as pure substances (6, 7) andas microparticles (CS-6, CS-7) were orally administered, once perday at the concentration 1/20 of LD50 as Tween 80 emulsion for aperiod of 21 days. On the 1st, 4th, 7th, 10th, 12th, 15th, 18th,21st day the blood glucose levels were measured using glucometer.At the end of the experiment, retro orbital blood collection wasperformed and the glycosylated hemoglobin was determined usingAutomatic Biochemistry Analyzer, Rx Imola.
The experiment was carried out in accordance with the currentguidelines for the veterinary care of laboratory animals and wereperformed with the consent of Ethics Committee for Animal
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Table 1The characteristics of chitosan microparticles.
CS Concentrationof CS solution(%)
Concentrationof TPP solution(%)
Particleformation
Particle size (lm)
Dry Wet
CSMMW 2 2 U 279 ± 3.4 566 ± 2.5CSMMW 2 5 U 263 ± 3.5 637 ± 3.8CSMMW 2 10 U+x 262 ± 3.1 701 ± 5.2CSMMW 1 2 U+x 752 ± 4.6 851 ± 4.5CSMMW 1 5 U+x 426 ± 3.1 743 ± 5.6CSMMW 1 10 U+x 408 ± 3.7 769 ± 5.8CSLMW 2 2 U+x 392 ± 4.5 722 ± 6.1CSLMW 2 5 U+x 364 ± 4.7 629 ± 5.9CALMW 2 10 U+x 214 ± 2.5 316 ± 4.6CSLMW 1 2 U+x 643 ± 3.5 859 ± 6.2CSLMW 1 2 U+x 605 ± 3.9 746 ± 5.8CSLMW 1 10 x – –
U: Stable beads; U+x: unstable beads; x: unstable beads.
Table 2The characteristics of chitosan microparticles loaded with xanthine derivatives (6, 7).
CS Conc. ofCS (%)
Conc. ofTPP (%)
Conc. of6 (mg/ml)
Conc. of7 (mg/ml)
Particleformation
Size (lm)
CSMMW 2 5 2.5 0.5 U 600–900CSMMW 2 5 5.0 2.5 U 600–900CSMMW 2 5 7.5 5.0 U 600–900CSMMW 1 5 2.5 0.5 U+x 600–900CSMMW 1 5 5.0 2.5 U+x 600–900CSMMW 1 5 7.5 5.0 U+x 600–900CSLMW 2 5 2.5 0.5 U+x –CSLMW 2 5 5.0 2.5 U+x –CSLMW 2 5 7.5 5.0 U+x –CSLMW 1 5 2.5 0.5 x –CSLMW 1 5 5.0 2.5 x –CSLMW 1 5 7.5 5.0 x –
U: Stable beads; U+x: unstable beads; x: unstable beads.
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Research of ‘‘Grigore T. Popa’’ University of Medicine and PharmacyIasi (no 291/2013).3. Results and discussion
3.1. Synthesis
The xanthine-thiazolidine derivatives (6, 7) were synthesizedaccording to the method described in our previous paper(Lupascu et al., 2013). First, through the esterification oftheophylline (1,3-dimeythylxanthine) sodium salt (1) (obtainedby reaction of theophylline with sodium in absolute methanol)with ethyl chloroacetate (1,3-dimethylxanthin-7-yl)ethylacetate (2) was obtained. This intermediary was reacted withhydrazine hydrate and the corresponding hydrazide (3) was trea-ted with 4-bromophenylisothiocyanate leading to 1-[2-(1,3-dimethylxanthin-7-yl)acetyl]-4-(4-bromophenyl)thiosemicarbazide(4). By cyclization of (4) with chloroacetyl chloride the correspond-ing thiazolidine-4-one was obtained (5). Finally, thethiazolidine-4-one derivative was condensed with4-hydroxybenzaldehyde and 4-chlorobenzaldehyde (Fig. 1).
The structure of the xanthine derivatives was proved by IR, 1HNMR and 13C NMR spectroscopy. The IR spectrum of compound 2confirm the presence of the functional methyl and methylenegroups through stretching vibration bands, narrow of mediumintensity at 1369 cm�1, respectively 1432 cm�1. The carbonyl(AC@O) from ester group is proved by a narrow absorption bandof medium intensity at 1755 cm�1. In the 1H NMR spectrum ofcompound 2 the protons of methylene group (NACH2A) appearas a singlet at 5.17 ppm. The protons of the ethyl group from the
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ester appear as multiplet (ACH2A) at 4.17 ppm and triplet(ACH3) at 1.21 ppm respectively. The carbons of the ester partwere identified at 61.69 ppm (C9) and at 14.56 ppm (C11). Thestructure of hydrazide (3) is confirmed by the appearance in theIR spectrum of the characteristic bands for ANHANH2A group at3438 cm�1, 3307 cm�1 and 3299 cm�1. The structure was stronglysupported by the disappearance of the proton signals characteristicof the ester group that was replaced by the hydrazide group. In theIR spectrum of the thiosemicarbazide derivative (4), the high andsharp specific absorbtion band of the thione group (C@S) wasobserved at 1130 cm�1. The valence vibration of CABr bond pro-duced a narrow absorption band of medium intensity at544 cm�1. In the 1H NMR spectrum, the aromatic protons res-onated as two doublet signals at 7.53 and 7.46 ppm. The aromaticcarbons appear in the 13C NMR spectrum in the range of119.15–136.19 ppm.
The structure of the thiazolidine-4-one (5) was confirmed bythe presence in IR spectrum of CAS bond at 810 cm�1, as anabsorption band of low intensity determined by the bond valencevibration. The protons of the methylene group from the thiazo-lidine ring appear as a singlet at 5.08 ppm, which confirms thecyclization of thiosemicarbazide with chloracetyl chloride. Thecyclization was also confirmed through the presence of the thiazo-lidine carbons: C10 (148.42 ppm), C11 (30.06 ppm) and C12
(119.15 ppm).The structure of the new benzylidene-thiazolidine-4-one
derivatives (6, 7) was confirmed by appearance in the IR spectraof the absorption band of medium intensity at 1598 cm�1 (6) and1594 cm�1 (7) due to the anti-symmetric stretching vibration ofthe cumulative double bond (C@C). The structure of these com-pounds was strongly confirmed by 1H NMR and 13C NMR spectraldata. The proton of the methine group (@CHA) from thebenzylidene-thiazolidine-4-one ring appears as a singlet at7.14 ppm (6) and 7.21 ppm (7) The carbon signals of thebenzylidene-thiazolidine-4-one ring were identified at 113.22(C11), 167.25 (C12); 139.63 (C13) for the compound 6 and 113.22(C11), 165.27 (C12), 139.63 (C13) for the compound 7.
3.2. Chitosan microparticles
Chitosan microparticles were prepared by the interactionbetween positively charged amino groups of chitosan and nega-tively charged counterion of TPP. Chitosan with a pKa of 6.3 is poly-cationic and presents NH3
+ sites when is dissolved in acetic acid.When sodium tripolyphosphate (Na5P3O10) is dissolved in waterit dissociates to give both hydroxyl and phosphoric ions. Sincethe cross-linking of chitosan it depends on availability of the catio-nic sites and the negatively charged species, it is expected the pHof TPP to have a significant role, intensifying the crosslinking pro-cess (Ko et al., 2002; Belcheva et al., 1995). In the present study, thepH value of TPP solution was 9.0. At this pH, both hydroxyl andphosphoric ions are present and may compete with each other tointeract with the NH3
+ of chitosan. The amino groups bind to thehydroxyl ions by deprotonation. In order to obtain stable chitosanmicroparticles with good physical parameters different type of chi-tosan and several concentrations (of chitosan and TPP) were used.The results are presented in Table 1. The most stable chitosanmicroparticles were obtained using chitosan medium molecularweight (CSMMW) in concentration of 2% in acetic acid and TPP 5%.
3.3. Characterization of chitosan microparticles loading with xanthinederivatives
The molecular weight of chitosan as well as the concentration ofthe chitosan solution have an important role on formation and sta-bility of the chitosan loaded microparticles. Related to the chitosan
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CS-6a CS-6b CS-6c
CS-7a CS-7b CS-7c
CS
Fig. 2. SEM micrographs of loaded chitosan microparticle: CS-6a, CS-6b, CS-6c, CS-7a, CS-7b, CS-7c, CS.
Fig. 3. Swelling degree of CS-6 and CS-7 series microparticles in distilled water (CS: chitosan, CS-6a: chitosan–xanthine derivative 6 (2.5 mg/ml), CS-6b: chitosan–xanthinederivative 6 (5 mg/ml), CS-6c: chitosan–xanthine derivative 6 (7.5 mg/ml), CS-7a: chitosan–xanthine derivative 7 (0.5 mg/ml), CS-7b: chitosan–xanthine derivative 7(2.5 mg/ml), CS-7c: chitosan–xanthine derivative 7 (5 mg/ml).
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low molecular weight it was observed that the microparticles donot form at concentration of 1%. At concentration of 2% themicroparticles are formed but they are not stable over time, forboth xanthine derivatives at all concentrations (Table 2). The samething was observed for chitosan medium molecular weight at con-centration of 1%. More stable were the loaded microparticles basedon chitosan medium molecular weight in concentration of 2%. Atthis concentration stable chitosan loaded microparticle weresuccessfully formed for both xanthine derivatives (6, 7) at allconcentrations (derivative 6: 2.5 mg/ml, 5 mg/ml, 7.5 mg/ml;derivative 7: 0.5 mg ml, 2.5 mg/ml, 5 mg/ml).
3.3.1. Particle size and morphologyThe size of the chitosan microparticles loaded with xanthine
derivatives increases with the concentration of the compounds,being in the range of 600–900 lm (Table 2). The scanning electronmicroscopy (SEM) showed that chitosan microparticles (CS) have aregular, spherical shape. Upon loading the xanthine compoundsthe shape of the microparticle gets deformed. In addition to thedeformation of shape, big cracks appeared on the surface of themicroparticles. The irregularity in shape and the number of cracksfurther increases with the concentration of compounds. The defor-mation of shape and the appearance of cracks in the morphology of
Fig. 4. Swelling degree of CS-6 and CS-7 series microparticles in simulated gastric fluid (CS: chitosan, CS-6a: chitosan–xanthine derivative 6 (2.5 mg/ml), CS-6b: chitosan–xanthine derivative 6 (5 mg/ml), CS-6c: chitosan–xanthine derivative 6 (7.5 mg/ml), CS-7a: chitosan–xanthine derivative 7 (0.5 mg/ml), CS-7b: chitosan–xanthine derivative7 (2.5 mg/ml), CS-7c: chitosan–xanthine derivative 7 (5 mg/ml).
Fig. 5. The XRD spectra of the xanthine derivatives (6, 7) and of the loaded chitosan microparticle – CS-6c: chitosan-xanthine derivative 6 (7.5 mg/ml), CS-7c: chitosan-xanthine derivative 7 (5 mg/ml).
Fig. 6. The FT-IR spectra of the chitosan (CS), xanthine compounds (6, 7) and of the loaded chitosan microparticle (CS-6a, CS-6b, CS-6c, CS-7a, CS-7b, CS-7c).
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the microparticle were evident for both xanthine derivatives, 6 and7 (Fig. 2).
3.3.2. Swelling degree (SD)The swelling experiments were performed in distilled water
knowing that the releasing of the active substance from the poly-mer matrix is depending of the water absorption rate. Becausethe polymeric systems have been developed for oral administra-tion, the swelling behavior was also studied in simulated gastric
fluid (SGF, pH 1.6). The swelling ratio of the loaded chitosanmicroparticle (CS-6 series, CS-7 series) was drastically decreasedin reference to chitosan microparticles (CS) in both media. In dis-tilled water chitosan microparticle showed a swelling ratio ofaround 120%, while the loaded chitosan microparticles (CS-6 seriesand CS-7 series) showed a ratio of about 40% (Fig. 3). Although theswelling capacity of the loaded chitosan microparticles wasimproved in SGF, it remained lower than that of chitosan micropar-ticles (Fig. 4). The chitosan microparticles loaded with compound 7
Table 3Loading efficiency of the xanthine derivatives (6, 7) into chitosan microparticles.
Type ofmicroparticles
Concentrationof CS solution(%)
Concentrationof TPP solution(%)
Conc. of xanthinederivatives(mg/ml)
LE(%)
CH-6a 2 5 2.5 83.63CH-6b 2 5 5.0 86.78CH-6c 2 5 7.5 90.89CH-7a 2 5 0.5 68.98CH-7b 2 5 2.5 83.23CH-7c 2 5 5.0 85.41
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(CS-7) showed a higher swelling degree in the SGF medium in com-parison to the ratio registered in distilled water, while no signifi-cant difference was observed in the CS-6 series. Also the swellingbehavior of CS-7 series in SGF was much better than the CS-6 ser-ies. The highest swelling degree was recorded for CS-7a(0.5 mg/ml) and it was 2.7 times higher than the value recordedin distilled water.
Regarding the time necessary to reach the equilibrium state, itwas observed that in distilled water the swelling equilibriumwas reached within 2 h whereas in the SGF, the microparticles stillshowed a swelling trend after 48 h.
The reduced swelling degree of the loaded chitosan microparti-cles (CS-6, CH-7) in comparison to the chitosan microparticles (CS)is due to the hydrophobic character of the xanthine derivatives (6,7) which reduce the swelling capacity of the polymer matrix.
3.3.3. X-ray diffraction analysisIn order to investigate the structure of the xanthine derivatives
(6, 7) and of the loaded chitosan microparticles (CS-6, CS-7) theXRD spectra were recorded. The compounds (6 and 7) have shownintense peaks in the range of 12–30� due to their highly crystallinecharacter (Fig. 5). The loaded chitosan microparticles (CS-6, CS-7)showed peaks of much lower intensity due the dispersion of thecompounds, at the molecular level, in the polymer matrix of chi-tosan microparticles. The peaks of CS-6c were more intense thanthose of CH-7c due to the higher amount of the compound presentin the polymer matrix: 7.5 mg/ml (CS-6c) in comparison to5 mg/ml (CS-7c).
3.3.4. Fourier transform infrared analysisThe presence of the xanthine derivatives (6, 7) in the polymer
matrix of chitosan microparticles has been proven by the FT-IRspectral data (Fig. 6). The spectra of chitosan micropaticles showedthe following characteristic bands: 1642 cm�1 (ACOANHANH2),
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Fig. 7. The loading efficiency (%) for CS-6 series: CS-6a (2.5 mg/ml); CS-6b (5 mg/ml);(5 mg/ml).
1420 cm�1 (CH2), 1025 cm�1 (CAOAC) and 1380 cm�1 (CH3). Thespectral bands at 1214 cm�1 (P@O) and 883 cm�1 (PAOAP) wererelated to the bands of crosslinking agent (TPP). The characteristicbands of the compound 6 were identified at 3234 cm�1 (ANHA),1639 cm�1 (AC@N), 1545 cm�1 (AC@O amide), 972 cm�1
(ACANA) cm�1. The bands at 3238 cm�1 (ANHA), 1642 cm�1
(AC@N), 1543 cm�1 (AC@O amide), 943 cm�1 (ACANA) and688 cm�1 (CACl) cm�1 are assigned to compound 7. In the IR spec-tra of CS-6 series and CS-7 series new bands characteristic to thexanthine-thiazolidine structure have appeared due to the presenceof this structure in the polymer matrix.
3.3.5. Loading efficiency (LE)The loading efficiency of the xanthine derivatives (6, 7) in the
chitosan microparticles is shown in Table 3 and Fig. 7. As it canbe observed, the amount of xanthine derivatives (6 and 7) thatwas loaded into the matrix of chitosan increased with the initialconcentration of the derivatives which were added in the chitosansolution. For xanthine derivative 6 at concentration of 7.5 mg/mlthe loading efficiency was 90.89% and for xanthine derivative 7at concentration of 5 mg/ml this parameter was 85.41%.ly
3.3.6. In-vitro releaseIt is known that the release of the active substance from a poly-mer support is a complex process that involves the passage of thesubstance through several biological media. In this study thereleasing profile of the xanthine derivatives (6, 7) loaded into chi-tosan microparticles was studied using three types of simulatedbiological fluids: simulated gastric fluid – SGF (pH 1.6), simulatedintestinal fluid – SIF (pH 6.5) and simulated colonic fluid – SCF(pH 6.5). The composition of the simulated biological fluids is pre-sented in Table 4.
The in-vitro (SGF, SIF, SCF) cumulative release profile was simi-lar for both loaded chitosan microparticles (the CS-6 series, theCS-7 series). A slower release was observed in the SGF medium(about 15%), a maximum release of the compounds occurred inthe SIF (50–75%) and relatively no release was observed in theSCF (Figs. 8 and 9). In the case of chitosan microparticles loadedwith compound 6 (CS-6), the system with the highest compoundconcentration (CH-6c) released 55.97% of the loaded compoundwhile microparticles with the lowest compound concentration(CH-6a) had the maximum release of 92.15% (Fig. 8).
A similar releasing profile was observed for chitosan micropar-ticles loaded with compound 7 (CS-7). The polymer system withlower concentration of the compound (CS-7a) released 91.33%while for CS-7c it was recorded a release of 68.65% (Fig. 9).
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CS-6c (7.5 mg/ml) and CS-7 series: CS-7a (0.5 mg/ml); CS-7b (2.5 mg/ml); CS-7c
Table 4The composition of the simulated biological fluids (SGF, SIF and SCF).
Composition SGF SIF SCF
Sodium taurocholate 80 lM 3 mM 10 mMLecithin 20 lM 0.2 lM 3 mMPepsin 0.1 mg/ml – –Maleic acid – 19.1 mM 28.6 mMSodium oleate – – 40 mMSodium hydroxide – 34.8 mM 52.5 mMSodium chloride 34.2 mM 68.6 mM 145.2 mMpH 1.6 6.5 6.5
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The release pattern of the both loaded chitosan microparticle(CS-6, CS-7) can be directly linked to their swelling characteristicsbecause the release rate from a polymer system is particularly areflection of its swelling behavior (diffusion mechanism).
The low releasing rate recorded in the SGF could be explainedby the hydrophobic character of the active compound (6, 7) whichlimits and delay the swelling process of microparticles and thusthe releasing rate of active compounds. When the loaded chitosanmicroparticles pass in the SIF they are swollen and thus theyrelease the largest amount of the loaded compounds. We supposethat the amount of the released compound in the SCF was rela-tively smaller and slower and thus it could not be detected byUV spectroscopy.
3.3.7. In vitro biodegradationIt is well know that chitosan is biodegraded by lyzozyme
through enzymatic depolymerization (Mawad et al., 2015).Lyzozyme can be found in a higher concentration in tear secretion,saliva, mucus, gastric fluid and mainly contributes to the initialdegradation rate of the polysaccharide by hydrolysis of the glyco-sidic bonds present in the macromolecular backbone. The chitosanenzymatic biodegradation is very important in the controlledrelease of active substance through polymeric membrane damage.The enzymatic biodegradation of chitosan was also proved in ourstudies, as it is shown in Fig. 10. It was observed that the stabilityof the loaded chitosan microparticles depends on the concentra-tion of the loaded compound. The chitosan microparticles loadedwith active substances (6, 7), in high concentration, showed anincreased biodegradation rate that could be explain by negativeinfluence of hydrophobic and crystalline state of the compoundsr P
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Fig. 8. The release profile of compound 6 from the loaded chitosan micropaticles(CS-6: CS-6a, CS-6b, CS-6c).
Fo
on stability of the polymeric systems. For CS-6c (7.5/mg/ml) thebiodegradation rate was 62.16% while for CS-6a (2.5 mg/ml) thebiodegradation was only 51.63% after 7 days. A similar biodegrada-tion rate was observed for CS-7 series, 58.09% for CS-7c (5 mg/ml)versus 45.60% for CS-7a (0.5 mg/ml). In similar experimental condi-tions the chitosan microparticles (CS) are more stable, after 7 daysthe biodegradation rate being of only 40.59%.
3.4. Biological evaluation
3.4.1. DPPH radical scavenging assayDPPH (2,2-diphenyl-1-picrylhydrazyl) is a well-known radical
which demonstrates a strong absorption band centered at about517 nm. It is scavenged by antioxidants through the donation ofproton forming the reduced DPPH with yellow color (Osorioet al., 2012). The scavenging activity of the tested compounds (6and 7) is presented in Fig. 11. It was observed that the maximumscavenging activity of the tested compounds appeared at 30 minand remained constant up to 40 min after starting the experiment.It was also observed that the substituent present on the phenylring had a great influence on the antioxidant activity. Compound6 which has 4-hydroxy on phenyl ring was more active than com-pound 7 which has 4-chlor on phenyl ring. After 40 min the DPPHremaining (%) was 41.78% in the case of compound 6, in compar-ison to 73.55% registered in the case of compound 7. Based onthese data it can be concluded that compound 6 is 1.8 times moreactive than compound 7 and 2.4 times more active than control (%DPPH remaining = 100).
3.4.2. Acute toxicity degreeThe determination of the LD50 is usually an initial step in the
assessment of the toxicity degree of the compounds. The resultsshowed that the tested compounds (6 and 7) have a lower toxicity,LD50 being 1625 mg/kg and 1594 mg/kg respectively. In referenceto theophylline (LD50 = 235 mg/kg) (Lindamood et al., 1987), usedas parent molecule in the synthesis of the compounds, the testedcompounds were approximately 7 times less toxic. In referenceto pioglitazone (LD50 = 181 mg/kg) (USP, 2010), a standard antidia-betic compound with thiazolidine structure, the tested compoundswere approximately 9 times less toxic.
3.4.3. Antidiabetic effects3.4.3.1. Blood glucose level. Chronic oral administration, for 21 days,of xanthine-thiazolidine derivatives (6 and 7) in an equivalent doseof 1/20 LD50 as compounds (6: group 3; 7: group 4) and as loadedchitosan microparticles (CS-6: group 1; CS-7: group 2) have
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Fig. 9. The release profile of compound 7 from the loaded chitosan micropaticles(CS-7: CS-7a, CS-7b, CS-7c).
Fig. 10. The enzymatic biodegradation profile of the loaded chitosan microparti-cles, CS-6 series: CS-6a (2.5 mg/ml); CS-6b (5 mg/ml); CS-6c (7.5 mg/ml) and CS-7series: CS-7a (0.5 mg/ml); CS-7b (2.5 mg/ml); CS-7c (5 mg/ml).
Fig. 12. Å jeun glucose level of the diabetic rats treated with CS-6 (group 1), CS-7(group 2), compound 6 (group 3), compound 7 (group 4), pioglitazone (group 5) inreference with diabetic (group 6) and non-diabetic rats (group 7).
Fig. 13. The blood glycosylated hemoglobin (HbA1c) level of diabetic rats treatedwith CS-6 (group 1), CS-7 (group 2), compound 6 (group 3), compound 7 (group 4),pioglitazone (group 5) in reference to diabetic (group 6) and non-diabetic rats(group 7).
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significantly reduced the blood glucose level in reference to control(untreated diabetic rats: group 6) (Fig. 12). The best hypoglycemiceffect was showed by compound 6 and chitosan microparticlesloaded with this compound (CS-6) which has hydroxy substituenton the benzylidene ring. At an equivalent dose of 1/20 DL50 thelowering of blood glucose level was 85.69% for CS-6 (after 15 days)and 49.30% for compound 6 (after 21 days) respectively. On the12th day, the lowering of the blood glucose level induced byCS-6 (a blood glucose level of 114.5 mg/dl) was even more intensethan that of pioglitazone (a blood glucose level of 148.5 mg/dl)used as standard antidiabetic drug. For CS-6 the experiment wasstopped on the 15th day because the hypoglycemic effect was veryintense (a blood glucose of 30 mg/dl) leading to death of the ratsthrough hypoglycemic shock.The hypoglycemic effect showed by xanthine-thiazolidinederivative 7 administered as compound (group 4) and as loadedchitosan microparticles (CS-7: group 2) was less than the effectof compound 6. On the 21th day the blood glucose level was low-ered by 35.68% (group 4, 375.6 mg/dl), respectively by 35.08%(CS-7, 361 mg/dl). In similar conditions pioglitazone reduced theblood glucose level at 167.5 mg/dl (group 5, 69.27%). rso
3.4.3.2. Glycosylated hemoglobin level. Chronic administration, ofxanthine-thiazolidine derivatives (6 and 7) in an equivalent doseof 1/20 LD50 as compounds (6: group 3; 7: group 4) and as loadedchitosan microparticles (CS-6: group 1; CS-7: group 2) was alsoassociated to a significantly reduction of the blood glycosylatedhemoglobin (HbA1c) level in reference to control (untreated dia-betic rats: group 6, HbA1c = 6.50%) (Fig. 13). The effect of com-pound 6 and its chitosan microparticles formulation (CS-6) wasmore intense than the effect of compound 7 and CS-7 respectively.On the 21th day the following level of HbA1c was recorded: 4.35%for group 1 (CS-6) and 4.30% for group 3 (compound 6). The effectFor Pe
Fig. 11. The scavenging activity of the xanthine-thiazolidine derivatives (6, 7).
was similar to the one of pioglitazone (group 6, 4.12%). In similarconditions the values of HbA1c recorded for compound 7 (group4) and CS-7 (group 2) were 5.20%, respectively 4.53%.
4. Conclusions
New xanthine derivatives (6, 7) as potential antidiabetic drugshave been synthesized and their structure was proved throughspectral methods (IR, 1H NMR, 13C NMR). Based on the beneficialeffects of chitosan as drug delivery system as well as its demon-strated antidiabetic effect new xanthine–chitosan formulationhave been developed in order to improve the pharmacokineticand pharmacological profile of the developed xanthine derivatives.The optimized formulations were evaluated in terms of particlesize, morphology, swelling degree, X-ray diffraction and Fouriertransform infrared analysis. The results demonstrated that theloading efficiency of the xanthine derivatives in chitosan micropar-ticles was between 68.98% and 90.89% depending of the type offormulation. The new developed formulation showed also anincreased cumulative release in the simulated biological fluids,SGF, SIF, SCF (between 55.97% and 92.10%) and a good biodegrada-tion rate (between 45.60% and 62.16%). Xanthine derivatives, espe-cially compound 6, showed a good antiradical scavenging effect(DPPH remaining 41.78%), reduced toxicity level (DL50 of1625 mg/kg) and improved antidiabetic effect. Chronic oral admin-istration of xanthine derivatives 6 on rats with streptozotocininduced diabetes mellitus was associated to a lowered level
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(49.30%) of the blood glucose, while the glycosylated hemoglobinlevel (4.30%) was similar to the one of pioglitazone (4.12%). Theantidiabetic effect of the xanthine derivative 6 was improved byloading in chitosan microparticles, the chitosan formulation,CS-6, showing an improved hypoglycemic effect. On the 12th dayof the experiment, the lower blood glucose level induced by CS-6(a blood glucose level of 114.5 mg/dl) was even more intense thanthe one induced by pioglitazone (a blood glucose level of148.5 mg/dl) used as standard antidiabetic drug. The level ofHbA1c was 4.35% in reference to the value of 4.12% recorded forpioglitazone. All these results demonstrate the potential applica-tion of xanthine derivative 6 and its chitosan formulation (CS-6)in the treatment of the diabetes mellitus syndrome.
Acknowledgments
This paper was published under the frame of European SocialFound, Human Resources Development Operational Programme2007-2013, project no. POSDRU/159/1.5/S/136893’’.
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