Research Article. p-coumaric acid regulates blood glucose and antioxidant levels in streptozotocin induced diabetic rats

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1 Aville online Journl of Chemil nd Phrmeutil Reserh, 215, 7(7): Reserh Artile ISSN : CODEN(USA) : JCPRC5 p-coumri id regultes lood gluose nd ntioxidnt levels in streptozotoin indued dieti rts Venktesn Amln, Ntesn Vijykumr* nd Arumugm Rmkrishnn Deprtment of Biohemistry nd Biotehnology, Annmli University, Annmli Ngr, Indi ABSTRACT Antihyperglyemi gents from nturl nd syntheti soures hve een found to suessfully mnge dietes. This study ws to hypothesis tht the orl dministrtion of p-oumri id regultes lood gluose nd ntioxidnt levels in streptozotoin (STZ) indued dieti rts. The STZ indued dieti rts exhiited inresed level of lood gluose, lowered the level of insulin, nd deresed the tivities of enzymti nd non enzymti ntioxidnts in lood s ompred with norml rts. Orl dministrtion of p-oumri id (1 mg/kg.wt) ws given for period of 3 dys. In our study we reognized tht the dministrtion of p-oumri id ould e lowered lood gluose nd improve the level of insulin, enzymti nd non enzymti ntioxidnts tivities in irultion. The histopthologil study of pnres, liver nd kidney reveled the protetive role of p-oumri id. We ould not oserve ny signifint hnges in ll the iohemil nd histopthologil studied in norml rts treted with p- oumri id. The overll results suggest tht the p-oumri id possesses potentil ntihyperglyemi nd ntioxidnt tivity in STZ-indued dieti rts. Keywords: p-oumri id, ntihyperglyemi, streptozotoin. INTRODUCTION Type 2 dietes mellitus (DM) onsists of rnge of dysfuntions hrterized y hyperglyemi nd resulting from the omintion of resistne to insulin tion, indequte insulin seretion, nd exessive or inpproprite glugon seretion this leds to hroni hyperglyemi [1]. During DM persistent hyperglyemi uses n inresed prodution of retive oxygen speies (ROS) vi uto-oxidtion of gluose nd non-enzymti protein glytion whih my led to disruption of ellulr funtions nd oxidtive dmge to memrnes [2]. An inrese in ROS is n impirment of ntioxidnt defense system or n insuffiient pity to repir oxidtive dmge [3]. Insulin ffets mny sites of mmmlin lipid metolism. It stimultes synthesis of ftty id in liver dipose tissue nd in the intestine. The insulin hs lso een reported to inrese the holesterol synthesis [4]. The tivity of lipoprotein lipse in white dipose is lso inresed. From this point of view the ssessment of vrious lipid frtions nd lipid peroxide in the ses of DM my e help in the prognosis of ptients nd in preventing the possiilities of omplitions or seondry disorders [5]. Peroxidtion of lipid memrne hs een relted to the pthogenesis of mny degenertive diseses, suh s theroslerosis, oxidtive dmge to DNA ging, rinogenesis, sikle ell disese nd et. [6]. In 213, there were 382 million people with dietes, nd this numer is expeted to rise to 592 million y 235 [7]. In Indi, it is estimted tht 61.3 million people with dietes ged 2-79 yers live (211 estimtes). This numer is 831

2 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): expeted to inrese to 11.2 million y 23 [8]. In 2, Indi (31.7 million) topped the world with the highest numer of people with dietes mellitus followed y Chin (2.8 million) with the United Sttes (17.7 million) in seond nd third ple respetively. Streptozotoin (STZ) is rod spetrum ntiioti nd lkylting genotoxi gent whih possesses ntiteril, tumoriidl, rinogeni nd dietogeni properties [9;1]. Indution of experimentl dietes in rts using streptozotoin is very simple to do nd provides onvenient model to study the tivity of hypoglyemi gents [11;12]. Furthermore, herl supplements nd other hoie of mediines hve progressively inrese to use for the tretment of dieti disorders [13]. Plnt derived polyphenoli ompounds possess wide rnge of phrmologil properties nd the study of their mehnism of tion hs een the sujet of onsiderle interest in reent yers [14]. World Helth Orgniztion (WHO) hs given suffiient stress in utilizing trditionl plnts nd plnt produts for dietes, sine they re non-toxi, effiient, with less or no side effet. There is n inverse ssoition etween dietry phenoli ompound intke nd mortlity from vrious diseses. Phenoli ompounds re group of phenoli ids tht re widely distriuted in whole grins, fruits, pers, vegetles nd everges suh s te, offee, wine nd hoolte [15]. p-coumri id (3-[4-hydroxyphenyl]-2-propenoi id) is phenoli ompound, undntly present in pinepple. p-coumri id is uiquitous plnt metolite possess ntioxidnt [16], ntiinflmmtory, ntiner [17], nd heptoprotetive effet [18]. In the present study, we ssess the effet of p-oumri id on the levels of gluose, insulin nd the tivities of enzymti nd non enzymti ntioxidnts nd lso the hnges ours in the histopthology of pnres, liver nd kidney tissues of experimentl rts. EXPERIMENTAL SECTION 1.1. Animls All the experiments were rried out with mle lino Wistr rts weighing 18-22g, otined from the Centrl Animl House, Rjh Muthih Institute of Helth Sienes, Annmli University, Tmil Ndu, Indi. They were housed in polypropylene ges (47 m 34 m 2 m) lined with husk, renewed every 24 h under 12:12 h light/drk yle t round 22 C nd hd free ess to tp wter nd food. The rts were fed on stndrd pellet diet (Prnv Agro Industries Ltd., Mhrshtr, Indi). The experiment ws rried out ording to the guidelines of the Committee for the Purpose of Control nd Supervision of Experiments on Animls (CPCSEA), New Delhi, Indi nd pproved y the Animl Ethil Committee of Annmli University (Approvl no. 175; dted 17/4/214) Chemils p-coumri id nd Streptozotoin (STZ) were purhsed from Sigm Chemil Co (St. Louis, Mo. USA). All other hemils nd solvents were of nlytil grde nd purhsed from Himedi Lortories Pvt. Ltd, Mumi, Indi Experimentl indution of type 2 dietes in rts Dietes ws indued in overnight-fsted experimentl nimls y single intrpertonil (i.p) injetion of STZ (4 mg/kg.w.), dissolved in itrte uffer (.1M, ph 4.5) [19]. The nimls were llowed to drink 2% gluose solution overnight to overome the initil drug-indued hypoglyemi mortlity. Control rts were injeted with sme volume of itrte uffer lone. After 72 h, plsm gluose ws determined nd those rts with fsting lood gluose greter thn 25 mg/dl were used in the present study Experimentl design In this study, totl of 24 rts were divided in to four groups of six rts eh. p-coumri id dissolved in.2% dimethyl sulfoxide (DMSO) nd dministered to rts orlly using n intr gstri tue dily for period of 3 dys. Group 1: Norml ontrol (vehile treted; DMSO: 1ml/kg.w.) Group 2: Norml + p-coumri id (1 mg/kg.w.) Group 3: Dieti ontrol (4 mg/kg.w.) Group 4: Dieti + p-coumri id (1 mg/kg.w.) At the end of the experimentl period, rts were fsted overnight nd srified y ervil dislotion. Blood smples were olleted in tues ontining potssium oxlte nd sodium fluoride (3:1) mixture used for the 832

3 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): estimtion of plsm gluose nd insulin. The pnres, liver nd kidney tissues were disseted nd olleted in ieold formlin for histopthologil estimtions Biohemil nlysis Plsm gluose level ws estimted y the method of trinder using ommeril kit [23]. Plsm insulin ws ssyed y ELISA kit (Boeheringer-Mnneheim Kit, Mnneheim, Germny). Thiorituri id retive sustnes (TBARS) nd hydroperoxides (HP) y the method of Niehius nd Smuelsson [45]. SOD [2], CAT [21]), GPx [22] Histopthologil exmintion Pnres, liver nd kidney were exised, fixed in 1% neutrl formlin, dehydrted in grded lohol (8-1%), lered in xylene, nd emedded in prf-169 fin. Then, the tissues were slied into 5 µm piees using mirotome, deprffinted in xylene, pssed through from 8% to 1% lohol nd ttined with hemtoxylin nd eosin for Olympus BX 4 photo-172 mirosope ssessments Sttistil nlysis All the dt were expressed s men + SD of numer of experiments (n= 6). The sttistil signifine ws evluted y one-wy nlysis of vrine (ANOVA) using SPSS Version 15 (SPSS, Cry, NC, USA) nd the individul omprisons were otined y Dunn s multiple rnge test (DMRT). Vlues re onsidered sttistilly signifint when P<.5 Dunn method (1957). RESULTS Effet of p-oumri id on plsm gluose nd insulin levels Figure 1 nd 2 desries the levels of plsm gluose nd insulin in different experimentl groups. Dieti rts exhiited inresed levels of plsm gluose with derese in insulin level when ompred to norml ontrol rts. Orl dministrtion of p-oumri id to dieti rts improved the glyemi sttus with signifint inrese in plsm insulin level. Figure-1. The hnges in the plsm gluose of ontrol nd experimentl rts Plsm gluose (mg/dl) Norml ontrol Control+poumri id Dieti ontrol Dieti+poumri id Groups Effet of p-oumri id on enzymti ntioxidnts in norml nd dieti rts Figure 3 nd 5 represents the tivities of ntioxidnt enzymes (SOD, CAT nd GPx) in erythroytes of experimentl nimls. A fll in the tivities of ntioxidnt enzymes were oserved in dieti rts when ompred to norml ontrol. The dministrtion of p-oumri id to dieti rts signifintly improved in the ntioxidnt sttus. 833

4 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): Figure-2. The hnges in the plsm insulin of ontrol nd experimentl rts Iinsulin (µu/ml) Norml ontrol Control+poumri id Dieti ontrol Dieti+poumri id Groups Figure-3. The hnges on the tivity of SOD in erythroytes of ontrol nd experimentl rts Enzyme onentrtion required to inhiit the NBT to 5% in one minute /mg H Norml ontrol Control+poumri id SOD Dieti ontrol Dieti+poumri id Figure-4. The hnges on CAT in erythroytes of ontrol nd experimentl rts µmole of H2O2 onsumed/minute/ mg H Norml ontrol CAT Control+poumri id Dieti ontrol Dieti+poumri id Effet of p-oumri id on non-enzymti ntioxidnts in norml nd dieti rts Figure 5 nd 6 shows the effet of p-oumri id on vitmin C, vitmin E nd GSH in plsm of ontrol nd dieti rts. In dieti rts the level of vitmin E, vitmin C nd GSH levels were deresed in the irultion. Orl dministrtion of p-oumri id inresed the vitmin E, vitmin C nd GSH levels when ompred with dieti rrs. 834

5 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): Figure-5. The hnges on GPx in erythroytes of ontrol nd experimentl rts GPx µg of GSH utilized/minute/mg H Norml ontrol Control+poumri id Dieti ontrol Dieti+poumri id Figure-5. The hnges on vitmin-c nd E in plsm of ontrol nd experimentl rts mg/dl Vitmin-C Norml ontrol Norml + p- oumri id Figure-6. The hnges on GSH in plsm of ontrol nd experimentl rts Vitmin-E Dieti ontrol Dietes + p- oumri id mg/dl GSH Norml ontrol Norml + p- oumri id Dieti ontrol Dietes + p- oumri id Effet of p-oumri id on histopthology of pnres, liver nd kidney Figure-7 to 9 shows histopthologil oservtion of pnres, liver nd kidney in experimentl rts fter 3 dys of tretment with p-oumri id (1 mg/kg.wt). H&E stining Histopthology studies of pnres of STZ indued dieti rt displyed redution of the Islets of lngerhns, dmged or redued the size of β-ells nd extensive nerosis hnges followed y firosis nd trophy. STZ indued dieti rt treted with p-oumri id restored the neroti nd firoti hnges nd rised the numer of β-ells. Histopthology studies of liver norml rts shows entrl vein surrounded y norml heptoytes. Dieti ontrol rts shows entrl vein, surrounded y dilted sinusoids with fol ftty hnges of heptoytes. p-coumri id treted rts present entrl vein surrounded y heptoytes. Histopthology studies of kidney Shows norml glomerulus with norml tuules. Dieti rt shows ongested glomeruli with lymphoyti in the interstium. p-coumri id treted rts present glomeruli surrounded y norml nd fin dilted tuules. 835

6 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): Figure-7. Histopthologil hnges in pnres of norml nd experimentl rts. All the setions re in H&E 1X () Norml pnres with islet ell surrounded y ini. () Norml + p-oumri id treted rt pnres. Norml rhiteture of pnreti ells. () Dieti rt pnres shows trophi ini with ftty infiltrtion. (d) Dieti + p-oumri id treted rt pnres. Preservtion of islet ells with few trophi ini. Figure-8. Histopthologil hnges of liver in norml nd experimentl rts. All the setions re in H&E 1X. () Norml ontrol liver showing the entrl vein nd heptoytes rrnged in the form of ords. () Norml liver treted with p-oumri id showing norml histology, () dieti liver showing fethery degenertion, miro nd mrovesiulr ftty hnges, periportl firosis nd vsulr ongestion, (d) dieti liver treted with p-oumri id hnges with the entrl vein ongestion. 836

7 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): Figure-9. Histopthologil hnges of kidney in norml nd experimentl rts. All the setions re in H&E 1X () Norml kidney showing glomeruli nd tuules. () Norml kidney treted with p-oumri id showing glomeruli nd tuules, () dieti kidney showing Glomeruloslerosis, vuotion in tuulr epithelil ells (d) dieti kidney treted with p-oumri id showing ner norml pperne of glomeruli nd tuules. DISCUSSION Glyolysis nd gluoneogenesis re the two primry omplementry events lning the gluose lod in our ody, whih is hrterized y prtil or totl defiieny of insulin nd plys pivotl role during the disrry of gluose metolism leding to elevted systemi gluose. Insulin prevents hyperglyemi, in prt, y suppressing hepti gluoneogenesis nd glyogenolysis nd filitting hepti glyogen synthesis [23]. STZ uses mssive redution in insulin relese, y the destrution of the β-ell of the islets of lngerhns nd therey indues hyperglyemi [24]. In dietes mellitus, insulin is not or insuffiiently synthesized, developing hyperglyemi with iohemil hnges in gluose nd lipid metolism leding to n inresed prodution of retive oxygen speies [25]. Fsting plsm gluose of dieti rts ws signifintly inresed thn norml rts. In p-oumri id treted dieti rts, signifint derese in lood gluose levels nd signifint inrese in plsm insulin levels were oserved in our erlier report [26]. No hnge ws oserved in the lood gluose levels of norml nd p-oumri id treted with norml rts. Phenoli ids suh s oumrin, innmi id et., stimulte the seretion of insulin in β-ells of pnres [27;28]. Norml mehnism of gluose indued insulin seretion egins with its trnsport into the β-ell y the GLUT-2 gluose trnsporter. Gluose phosphoryltion y gluokinse is the rte limiting step tht ontrols gluoseregulted insulin seretion. Further metolism of gluose-6-phosphte vi gyolysis genertes ATP, whih inhiits the tivity of nd ATP-sensitive K + hnnel. Inhiition of this K + hnnel indues β-ell memrne depolriztion, whih open voltge-dependent lium hnnels (leding to n influx of lium), nd simultes insulin seretion. The derese in lood gluose nd inrese the level of insulin in dieti rts treted with p-oumri id might e due to enhned insulin seretion nd inrese the utiliztion of gluose. The ntioxidnt enzyme system is losely integrted nd the ltertions of ny one enzyme my hve its effet on other enzymes [29]. Free rdil svenging enzymes suh s SOD, CAT nd GPx re the first line of ellulr defense ginst oxidtive injury whih is involved in the disposl of superoxide nions nd H 2 O 2 [3]. 837

8 Ntesn Vijykumr et l J. Chem. Phrm. Res., 215, 7(7): The enzymti ntioxidnts re plying vitl role in preventing ells from eing exposed to oxidtive dmge. SOD is ple of reduing the super oxide rdil in H 2 O 2. The other enzymti ntioxidnt CAT tlyzes the redution of hydrogen peroxides nd protets the tissues ginst retive hydroxyl rdils [31]. In dietes mellitus, high gluose n intivte ntioxidnt enzymes SOD, CAT nd GPx y glyting these proteins thus produing indued oxidtive stress, whih in turn, use lipid peroxidtion [32]. CAT, SOD nd GPx tivities were ought to ner norml inditing the effiy of p-oumri id in ttenuting the oxidtive stress in liver of dieti rts. Previous studies hve lso shown tht phenoli ompounds hd free rdil svenging properties nd redued the oxidtive stress ssoited with dietes mellitus [33]. Hene, ompound tht ould prevent the genertion of these oxygen free rdils or inrese the free rdil svenging enzymes my e effetive in STZ-indued dietes. In our study the enzymti ntioxidnt tivities suh s SOD, CAT nd GPx were deresed in dieti rts nd dieti rts treted with p-oumri id shows signifint inrese in these enzyme tivities. The results suggest tht p-oumri id possess free rdil svenging tivity, whih ould exert enefiil effet ginst pthologil ltertions used y free rdils. Non-enzymti ntioxidnts suh s vitmin C nd Vitmin E nd GSH, ply n exellent role in preventing the ells from oxidtive stress. Vitmin C (sori id) is hydrophili ntioxidnt nd disppers fster thn other ntioxidnts on exposure to ROS [34]. The deresed level of sori id in dieti rts my e due to inresed utiliztion of ntioxidnts ginst inresed ROS. The redution in Vitmin C levels my lso due to derese in glutthione level, sine glutthione is required for the reyling of sori id [35]. In our study, Vitmin C ws deresed in dieti rts s reported erlier [36]. Tretment with p-oumri id to dieti rts reversed Vitmin C to ner norml level whih ts s strong superoxide rdil nd singlet oxygen quenher. Vitmin E is one of the mjor hin reking lipophili ntioxidnts within the ell memrne where it protets memrne ftty ids from LPO. Vitmin E quenhes the singlet oxygen nd is onverted to vitmin E rdil. Vitmin E lso rets with lipid peroxides to terminte the rdil hin retion in the memrne lipids [37]. The deresed level of α-toopherol oserved in the dieti rts my e due to inresed utiliztion of vitmin E in svenging the oxyrdils generted y high levels of gluose (or) might e due to deresed vitmin C onentrtion euse there is well estlished synergism etween vitmin E nd vitmin C [38]. Deresed vitmin E levels hve lso een found in dieti ptients with inresed lipid peroxidtion produts [39]. Upon the tretment with p-oumri id to dieti rts inresed Vitmin E to ner norml rts whih my t s n inhiitor for further free rdil indued lipid peroxidtion. GSH is mjor endogenous ntioxidnt whih ounterlne free rdil medited dmge. It is involved in the mintenne of norml ell struture nd funtion, proly through its redox nd detoxifition retion [4]. GSH funtions s free rdil svenger nd in the repir of free rdil used iologil dmge [41]. GSH is required for the reyling of vitmin C nd ts s sustrte for GPx nd GST tht re involved in preventing the deleterious effet of oxygen rdils [42;34]. STZ-dieti rts exhiited deresed level of GSH whih might e due to inresed utiliztion for svenging free rdils nd inresed onsumption y GPx nd GST. Tretment with p-oumri id signifintly improved GSH level in plsm nd tissues of dieti rts whih ould e due to deresed lipid peroxidtion. Thus, p-oumri id exerts potentil ntioxidnt property, s evidened y inresed ntioxidnts sttus. Histopthologil exmintion of dieti pnres expressed islnd of islet ells with ftty infiltrtion. Administrtion of p-oumri id showed onserved islet ells with ini. Dieti liver expressed entrl vein, surrounded y dilted simusoids with fol ftty hnge of hepotoytes. Administrtion of p-oumri id expressed entrl vein surrounded y heptoytes. Dieti kidney showed ongested glomeruli with lymphoyti infilterte in the interstium. p-coumri id treted dieti kidney showed norml glomeruli free dilted tuules. Histopthologil oservtions suh s pnres, liver nd kidney re represented on Figure 7 to 9. CONCLUSION It ould e onluded tht tretment with p-oumri id (1 mg/kg.wt) exhiited preventive effets on ltered levels of lood gluose nd insulin. It is lso prevented enzymti nd non enzymti ntioxidnts. The possile mehnism for the oserved preventive effets of p-oumri id is due to its hypoglyemi nd free rdil svenging properties. Aording to our study p-oumri id my e enefiil for protetion ginst dietes nd its omplition. 838

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