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1 Kumr et l. SpringerPlus 213, 2:639 SpringerOpen Journl RESEARCH Open Aess Enhned glyemi ontrol, pnres protetive, ntioxidnt nd heptoprotetive effets y umelliferon-α-d-gluopyrnosyl-(2 I 1 II )-α-dgluopyrnoside in streptozotoin indued dieti rts Viks Kumr 1*, Dnish Ahmed 1, Firoz Anwr 2, Mohmmed Ali 3 nd Mohd Mujee 3* Astrt Ojetive: The ojetive of the present study ws to evlute the effet of umelliferon-α-d-gluopyrnosyl- (2I 1II)-α-D-gluopyrnoside (UFD) from Aegle mrmelos Corr. on serum gluose, lipid profile nd free rdil svenging tivity in norml nd STZ (streptozotoin) indued dieti rts. Mterils nd methods: Dietes ws indued y single interperitonel injeting of streptozotoin (6 mg/kg, i.p.) in the rts. All the rts were divided into following groups; I - nondietei, II - nondieti + UFD (4 mg/kg, p.o.), III - dieti ontrol, IV - UFD (1 mg/kg, p.o.), V - UFD (2 mg/kg, p.o.), VI - UFD (4 mg/kg) nd VII - glienlmide (1 mg/kg, p.o.). Serum gluose level nd ody weight were determined periodilly. Biohemil prmeter, ntioxidnt enzyme nd histopthology study were performed on the dy 28. Orl gluose tolerne test study ws performed to identify the gluose utiliztion pity. Results: All the doses of UFD nd glienlmide derese the level of serum gluose, glyted hemogloin, gluose-6-phosphtse, frutose-1-6-iphosphte nd inresed the level of plsm insulin, hexokinse. The UFD doses lso showed effets on ntioxidnt enzymes viz. superoxide dismutse, tlse nd glutthione peroxidse whih were signifintly inresed nd the level of mlonldehyde ws mrkedly deresed. Histologilly study, fol nerosis, deposition of fts, inresed the size of the interlted dis were oserved in the dieti rt liver, kidney, hert nd pnres ut ws less ovious in treted groups. The mehnism of tion of the UFD emerges to e due to inrese the tivity of ntioxidnt enzyme nd seretion of pnreti insulin. Conlusion: Redution in the FBG (fsting lood gluose), glyted hemogloin, gluose-6-phosphtse, frutose-1-6-iphosphte, superoxide dismutse, tlse, glutthione peroxides, holesterol, triglyeride, LDL, VLDL levels nd improvement in the level of the plsm insulin, hexokinse, HDL ws oserved y the UFD treted rts. The result indites tht UFD hs nti-dieti tivity long with nti hyperlipidemi nd ntioxidnt effiy nd provides sientifi rtionle to e used s n Anti-dieti gent. Keywords: Umelliferon-α-D-gluopyrnosyl-(2I 1II)-α-D-gluopyrnoside; Streptozotoin; Antidieti; Antihyperlipidemi; Glienlmide * Correspondene: phviks@gmil.om; mohdmujee72@gmil.om 1 Deprtment of Phrmeutil Sienes, Fulty of Helth Sienes, Sm Higginottom Institute of Agriulture, Tehnology & Sienes, Allhd, Uttr Prdesh 2117, Indi 3 Deprtment of Phytohemisty & Phrmognosy, Fulty of Phrmy, Jmi Hmdrd, New Delhi 1162, Indi Full list of uthor informtion is ville t the end of the rtile 213 Kumr et l.; liensee Springer. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 Kumr et l. SpringerPlus 213, 2:639 Pge 2 of 2 Figure 1 Struture of UFD. Introdution Dietes mellitus (DM) is group of syndrome hrterized y dietry intke, hnging in the lifestyle, exessive use of lipid, rohydrte nd protein. Poorly ontrolled lood gluose level is the mjor ftor in the development Tle 1 13 C NMR spetrl dt for ompounds BG II (UFD) Cron (Position) δ 1H (J in Hz) 13 C NMR (DMSO d 6 ) I I I I I I II II II II II II Coupling onstnts in Hertz re provided in prenthesis. of oth dieti omplition suh s type 1 dietes nd type 2 dietes (Amerin Assoition of Dietes Edutors 22). STZ is minly used for indution of experimentl utoimmune dietes. Low dose dministrtion of STZ in the peritonel vity of n niml is the est model for type I dietes. Orl hypoglyemi gents (insulin, sulphonylures, thizolidiones nd iogunides) nd different plnt sed drugs were used for the tretment of dietes, ut orl hypoglyemi drug hving some limittion in the tretment of dietes (Vlithn 1998). The plnts sed drugs re gining populrity dy y dy. These plnt sed drugs possess tive ingredient nd t on vriety of trgets y vrious mode nd mehnism. Severl speies of plnts hve een reported in the reputed lterntive system of mediine s est hoie for the tretment of dietes euse plnt sed ntidieti drug re onsidered less toxi nd free from side effets. The mjor drwk of the nturl therpy is limittion of iotive ompound for liming their ntidieti effet (Morin 1987). Most of the reserhers limed tht dietes omplitions were ourred y oxidtive stress (Hlliwell nd Gutteridge 1989). Clinil nd experimentl ondition of dietes inresing the level of oxidtive stress otherwise hnges in ntioxidnt pity nd produed the etiology of hroni dietes (Rvi et l. 24). Coumrins widely onsumed in the humn diet in the form of vegetle nd fruits (Hoult nd Py 1996), oumrins present in the food nd vegetle ply n importnt role s dietry ntioxidnts. Mny investigtor lim tht severl phenoli oumrins might ply role s dietry ntioxidnts, euse severl fruit nd vegetle were onsumed y humn eings s food. Aegle mrmelos Corr. (Rutee) is very ommon plnt found espeilly in hills of the Himly, dry forest nd

3 Kumr et l. SpringerPlus 213, 2:639 Pge 3 of 2 Tle 2 Effet of UFD on orl gluose tolerne test S. Time (min) No Gluose Control 81.6 ± ± ± ± ± ± UFD (1 mg/kg) 82.4 ± ± ± ± ± ± UFD (2 mg/kg) 81.6 ± 1.77 ns 13.4 ± ns 121 ± * 112 ± ** 97.4 ±.927 *** 84.6 ± *** 4 UFD (4 mg/kg) 82.2 ± ns ± 1.53 ** 112 ± *** 99.6 ± 1.28 *** 82 ± 2.1 *** 62.6 ± *** 5 Glienlmide (1 mg/kg) 81 ± ns 125 ±.77 ** ±.59 ** 13 ±.717 *** 85.8 ± *** 69.6 ± *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non signifint; ANOVA, followed y Dunnett s multiple omprison test. south Indi with ltitude (25 12 m) (Hjr et l. 1997; Gupt nd Tndon 24). Different prts (leves, fruit, rk nd stem) of the plnt re used s ethnomediine ginst fevers, domen pin, plpittion of the hert, urinry troules, melnholi, norexi, dyspepsi, dietes nd dirrhe (Bdm et l. 24; Gupt nd Tndon 24). More thn 1 hemil onstituent were isolted from the Aegle mrmelos Corre inluding eugenol, lupeol, egeline, mrmsinin, mrmin, skimminine, egelin, lupeol, ineole, itrl, itronelll, uminldehyde (4-isopropylenzldehyde), eugenol, mrmesinin, mrmelosin, luvngetin, urpten, psorlen, mrmelide, fgrine, nd tnnins. These hemil onstituents hve een proved tive ginst vrious disese like mlri, gstrointestinl nd ner disese. Different solvent extrts showed effetiveness ginst ntiuler, ntidieti, ntioxidnt, ntihyperlipidemi, ntipyreti, ntiinflmmtory on vrious models of niml. But the iotive ompound present in this extrt ws not identified in their nturl proess. Presently, there is no pulished soure for the lim out the ntidieti, ntihyperlipidemi nd ntioxidnt tivities of iotive ompounds isolted from A. mrmelos (Lithfield nd Wiloxon 1949; Mity et l. 29). Umelliferon-α-D-gluopyrnosyl-(2I 1II)-α-D-gluopyrnoside (shown in Figure 1), gluosidi derivtive of oumrin (6-hydroxyoumrin), isolted from the stem rk of A. mrmelos is powerful ntioxidnt. The present study investigtes the effet of orl dministrtion of iotive ompound, umelliferon-α-d-gluopyrnosyl-(2 I 1 II )- α-d-gluopyrnoside on ntidieti, ntihyperlipidemi nd ntioxidnt effet on STZ-indued dieti rts. Mteril nd methods Generl Veego, Model No. MPI melting point pprtus ws used for melting point. 1 H NMR spetr were reorded on Bruker Advne II 4 NMR Spetrophotometer nd 13 C NMR spetr on Bruker Advne II 1 NMR Spetrophotometer in DMSO using TMS s internl stndrd. Mss spetr were otined on the VG-AUTOSPEC spetrometer. UV λmx (DMSO) were reorded on Shimdzu UV-17 nd FT-IR (in 2. m-1, flt, smooth, Aex) were tken on Perkin Elmer Spetrum RX-I spetrophotometer. Chemil Streptozotoin (Sigm Chemil Co. USA), GOD/POD kit, Cholesterol kit, Triglyeride kit, (Spn, Indi), Glienlmide (Rnxy, Indi), Croxyl methyl ellulose (CMC) (SD fine, Indi) were purhsed from respetive vendor. Sili gel (6 12 mesh) (Nihols Indi Pvt. Ltd) ws used for olumn hromtogrphy. The entire regent utilized for experimentl protool nd hromtogrphi isoltion were of nlytil grde nd used without further purifition. Plsm gluose (mg / dl) Time (min) *** *** *** *** *** *** *** *** OGTT (mg/dl), Gp I, Norml Control OGTT (mg/dl), Gp II, UFD I (1 mg/kg) OGTT (mg/dl), Gp III,UFD (2 mg/kg) OGTT (mg/dl), Gp VI, UFD III (4 mg/kg) OGTT (mg/dl), Gp V, Glienlmide (1 mg/kg) Figure 2 Effet of UFD on fsting plsm gluose on orl gluose tolerne test t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; *P <.5; **P <.1; ***P <.1; P >.5 is onsidered s non-signifint (ns).

4 Tle 3 Effet of UFD on iohemil prmeter in STZ indued dieti rts S. No. Biohemil prmeter Norml ontrol Norml ontrol + UFD (4 mg/kg) STZ-dieti ontrol STZ dietes + UFD STZ dietes + UFD STZ dietes + UFD STZ dietes + Glienlmide (1 mg/kg) (2 mg/kg) (4 mg/kg) (1 mg/kg) 1 Fsting plsm gluose (mg/dl) 83.4 ± ± ± *** ± *** 143 ± 3.82 *** 17.4 ± *** ± *** 2 Fsting plsm insulin (μu/ml) 12 ± ± ±.374 *** 4.4 ±.59 * 7.2 ±.374 ** 1.4 ±.519 *** 1.2 ±.374 *** 3 Glyted hemogloin (A1) (%) 1.3 ± ± ±.17 *** 3.9 ±.172 * 3.2 ±.78 ** 2.34 ±.93 *** 2.56 ±.75 *** 4 Totl holesterol (mg/dl) 65.8 ± ± ± *** 11.6 ± * 82 ±.771 ** 68.4 ± *** 72.4 ± *** 5 Triglyerides (mg/dl) 83 ± ± ± *** ± * 19.2 ± 1.81 ** 91.6 ± *** 95.8 ± *** 6 HDL holesterol (mg/dl) 54.8 ± ± ± *** 37 ± 1.34 * 42.8 ± ** 53.2 ± *** 5.6 ± 1.77 *** 7 LDL holesterol (mg/dl) 11.4 ± ± ±.59 *** 74.8 ±.374 * 49 ±.316 ** 21.4 ± *** 27 ±.316 *** 8 VLDL holesterol (mg/dl) 16.6 ± ± ±.849 *** ±.344 * ±.361 ** ±.463 *** ±.471 *** 9 Hexokinse (μg/mg of tissue) ± ± ± *** 112 ± * ± ** ± *** ± *** 1 Gluose-6-phosphtse (unit/mg of tissue) 11 Frutose-1-6-iphosphtse (unit/mg of tissue) 9.2 ± ± ±.582 *** 13.6 ±.41 ns 11.4 ±.59 * 9.8 ±.374 *** 1.8 ±.372 *** 29.6 ± ± ± 1.77 *** 45.4 ±.927 * 35 ±.77 ** 25.6 ±.59 *** 29 ±.77 *** 12 Weight vrition (g) 22.2 ± ± ± 3.11 *** ± *** ± *** 23.4 ± *** 2.4 ± *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non signifint; ANOVA, followed y Dunnett s multiple omprison test. Compred to vehile ontrol. Compred to dieti ontrol. Kumr et l. SpringerPlus 213, 2:639 Pge 4 of 2

5 Kumr et l. SpringerPlus 213, 2:639 Pge 5 of 2 Mteril The stem rk of Aegle mrmelos Corre. ws olleted from the Botnil Grden, Deprtment of Phrmeutil Sienes, Fulty of Helth Sienes, Sm Higginottom Institute of Agriulture, Tehnology & Sienes Deemed University, Allhd, Uttr Prdesh, Indi nd uthentited y Dr. Imrn Kjmi (Phrmognosist). A speimen vouher (SIP/HD/54/12) of the plnt smple respetively hd een deposited in the herrium of Siddhrth Institute of Phrmy, Dehrdun, Uttrkhnd, Indi. Extrtion nd isoltion The shde dry stem rk of Aegle mrmelos Corre (2 kg) ws extrted with methnol (5 L) t 45 C for 72 hr. After extrtion totl filtrte ws onentrted to dryness in rottory vuum evportor t 4 C to otin uniform slurry (322 gm) (Kumr et l. 29; Kumr et l. 211; Kumr et l. 213). The slurry ws dissolved in smll mount of methnol nd sored on sili gel (6 12 mesh). It is sujeted to olumn using s C 6 H 14 /CHCl 3 /MeOH grdient system (1::, 2::, 4::, 4:1:, 1:1:, 1:4:, 1:6:, :1:, :48:, :24:1, :48:2, :1:, :1:1, :24:7, nd :47:1; 3. L for eh grdient system), yielding 22 frtions. The olleted frtions spotted on pre oted sili gel TLC plte nd the frtions hving the sme R f vlue pooled together in 7 frtions. Frtion 8 14 (13.5 g) were omined seprted on sili gel olumn (CHCl 3 /MeOH, 3:1), nd rehromtogrphed on sili gel olumn (CHCl 3 /MeOH, 8:1), yielding 3 sutrtions. Compound ws seprted y norml phse sili gel olumn (CHCl 3 /MeOH, 1:4). The ompound ws found to e 1% pure y HPTLC y using solvent system CHCl 3 /MeOH (2:1), see Figure 1. Drugs solution UFD nd glienlmide were emulsified with 2% roxyl methyl ellulose (CMC) dissolved in distilled wter. Streptozotoin ws dissolved in freshly prepred itrte uffer (ph = 4. 5). Animls Mle lino rt (Wistr strin 15-2 g) ws used for the experiment. The nimls were housed under stndrd onditions of temperture (25 ± 1 C), reltive humidity (55 ± 1%), 12 hr/12 hour light/drk yles nd fed on stndrd pellet diet (Lipton rt feed, Ltd., Pune) nd wter d liitum. The experimentl protool ws pproved y the Institutionl Animl Ethil Committee of Siddhrth Institute of Phrmy (1435/PO//11/CPCSEA). Aute toxiity study The toxiity studies were dopted s per OESD Guideline No.42; (Annexure-2d) of CPCSEA. For ute toxiity studies in norml helthy rts fsted overnight nd rndomly divided into five groups nd eh group ontin rts (n = 1). Rts were treted with strting doses (.5,.1,.5 nd.1 g/kg ody weight) of test ompound nd the ontrol group ws treted with vehile lone (CMC 2%; 1 ml/kg ody weight). All the niml groups llowed for food nd wter d liitum nd were followed over period of 2 h for hnging in vrious eonomil (Defetion nd urintion), neurologil (Spontneous tivities, retivity, touh response, pin response nd git) nd ehvior (Alertness, restlessness, irritility, nd ferfulness) responses (Lithfield nd Wiloxon 1949; Lipnik et l. 1995). The mortlity used y the extrt within this period of the time ws oserved. Assessment of ompound in n orl gluose tolerne test (Bonner-weir 1988) Helthy rts were divided into five groups of six nimls eh, Group I (Control): treted with vehile only. Group II (UFD): treted with ompound 1 mg/kg. Fsting plsm gluose (mg/dl) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 3 Effet of UFD on fsting plsm gluose t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

6 Kumr et l. SpringerPlus 213, 2:639 Pge 6 of 2 Body Weight (gm) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 4 Effet of UFD on ody weight t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). Group III (UFD): treted with ompound 2 mg/kg. Group IV (UFD): treted with ompound 4 mg/kg. Group V (Stndrd): treted with glienlmide 1 mg/kg. All group nimls reeived drug nd vehile orlly. After 3 min tretment with different doses of UFD nd glienlmide, ll groups rt reeived 2 gm/kg of gluose. The lood smple olleted from the retro-orit of the eye of rts t regulr intervl of, 3, 6, 9, 12 nd 15 min eh for their gluose tolerne. Indution of dietes Dietes ws indued in the Wistr rts y using the single interperitonel injetion of streptozotoin (6 mg/kg ody weight). Volume of (STZ) 1 ml/kg ody weight prepred y STZ dissolving in freshly prepred.1 M itrte uffer (ph = 4.5) (Brosky nd Logothelopoulos 1969; Ahmed et l. 213). After 3 dy of STZ dministrtion, lood gluose level of rts ws estimted. Rts with lood gluose level of 27 mg/dl eyond were onsidered s dieti. Experimentl design nd shedule The rts were rndomly divided into 7 groups nd eh group ontins 6 nimls. Group I (Norml Control): Untreted group Group II (Norml Control): UFD 4 mg/kg Group III (Dieti Control): Untreted group Group IV: treted with ompound UFD 1 mg/kg Group V: treted with ompound UFD 2 mg/kg Group VI: treted with ompound UFD 4 mg/kg Group VII: treted with glienlmide 1 mg/kg. The tretment ontinued for 28 dys y dministrtion of different doses of UFD nd glienlmide suspended in.2% CMC one dily (Nihols 1956). The fsting lood gluose level ws determined dy, 5, 1, 15, 2, 25 nd 28th dy. During the experiment period hnge in the ody weight of rt ws lso reorded. Estimtion of iohemil prmeter Theloodsmpleswerewithdrwnonthedy28olleted from retro oritl punture tehnique y pillry tues Plsm insulin (µu/ml) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 5 Effet of UFD on level of plsm insulin t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

7 Kumr et l. SpringerPlus 213, 2:639 Pge 7 of 2 Glyerided Hemogloin (A1) (%) ns Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 6 Effet of UFD on level of glyted hemogloin (A1) (%) t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). ontining ntiogulnt (disodium ethylene dimine tetr ette) under mild nesthesi; lood ws entrifuged nd exmined for plsm gluose nlysis ws done y GOD - POD method using the Gluose Estimtion Kit (Spn Dignosti, Indi). Other serum estimtion ws done spetrophotometrilly using stndrd kits whih inlude totl holesterol, HDL nd triglyeride (Spn Dignosti, Indi). Plsm insulin ws estimted y the method of reported method of (Nihols 1956). For determintion of the ntioxidnt enzyme, liver ws homogenized in ie hilled 1% potssium hloride solution for estimting different prmeters viz. superoxide dismutse (SOD), tlse (CAT), glutthione peroxidse (GPx) nd mlonldehyde (MDA) (Sinh 1972; Rotruk et l. 1973; Kkkr et l. 1984). Histopthology For histopthology study, fter 28 dys ll group nimls were srified under mild nesthesi nd different orgns (hert, liver, pnres nd liver) were isolted for histopthologil nlysis. The isolted orgn tissue ws fixed t 1% nturl uffered formlin, dehydrted y pssing through grded series of lohol, nd emedded in prffin loks nd 5 mm setion ws prepred using semi-utomted rottory mirotome. Hemtoxylin nd eosin were used for stining. Results Compound identifition The methnoli extrt of dried stem rk powder of A. mrmelos ws sujeted to olumn hromtogrphy. Chromtogrphilly identil frtions (hving the sme R f vlues) were mixed together nd onentrted. Colleted frtions were further purified y sili gel reolumn hromtogrphy to isolte ompound BG II (5 mg). ESI-MS t m/z (rel. int.): 486 [M] + C 21 H 26 O 13 (2.2), 1 H NMR (DMSO-d 6 ): Tle 1; 13 C NMR (DMSO-d 6 ): Tle 1; IR λmx (KBr): 3452, 341, 3325, 2929, 2848, 172, 1629, 1515, 1457, 1384, 127, 1118, 151 m-1, UV λmx 256, 277, 332 nm (log ε 4.1, 5.8, 3.1) (Additionl file 1: Spetrl Dt of umelliferon-α-d-gluopyrnosyl-(2 I 1 II )-α-dgluopyrnoside). Hexokinse (µg/mg of tissue) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 7 Effet of UFD on level of Hexokinse t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

8 Kumr et l. SpringerPlus 213, 2:639 Pge 8 of 2 Gluose-6-Phosphtse (Unit/mg of tissue) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 8 Effet of UFD on level of Gluose-6-phosphtse t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P<.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). Effet of UFD on ute toxiity Aute toxiity studies exposed the non-toxi nture of the isolted ompound UFD. During the ute toxiity study of the UFD on Wistr rts no mortlity nd no hnge in the ehvior were oserved t end of the study. There ws no lethlity or toxiity found t ny seleted doses until the end of the study. Effet of UFD on orl gluose tolerne test The orl gluose tolerne test ws evluted in overnight fsted rts. The effet of different doses of UFD on orl gluose tolerne presented in the Tle 2. The strting gluose level of the overnight fsting rt ws 81.6 mg/dl. Wistr rts fter treted with gluose the levels of lood gluose inresed were oserved. Tretment ws initited with different doses of UFD nd glienlmide signifintly redued the lood gluose level t 15 min nd normlize ner to norml ontrol group rt (Figure 2). Signifintly, diminishing level of lood gluose ws oserved with UFD dose 1 mg/kg (28.71%), UFD 2 mg/kg (35.12%), UFD 4 mg/kg (48.43%) nd glienlmide 1 mg/kg (44.32%). Effet of UFD on lood sugr level Administrtion of STZ produed the dietes, therey inrese the lood sugr level. The lood gluose level of norml ontrol group rts ws 83.4 mg/dl. In the STZ dieti rts, the level of lood gluose rehed to mg/dl t dy 28. STZ indued dieti rts treted with different doses of the UFD nd glienlmide showed the signifintly lowered the lood gluose level till 28 dys. Seleted doses of UFD 1, 2 nd 4 mg/kg nd glienlmide 1 mg/kg lowered the lood gluose level y 4.84%, 52.33%, 63.45% nd 6.45% respetively, thus showing signifint derese in lood gluose level (Tle 3, Figure 3). Frutose 1-6-iphosphte (unit/mg of tissue) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 9 Effet of UFD on level of Frutose1-6-iphosphte t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

9 Kumr et l. SpringerPlus 213, 2:639 Pge 9 of 2 Totl Cholesterol (mg/dl) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 1 Effet of UFD on level of totl holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P<.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). Effet of UFD on ody weight The initil ody weight ws similr in non dieti s well s dieti ontrol groups. The dministrtion of different doses of UFD nd glienlmide treted group signifintly (P <.1) prevented derese in ody weight (Tle 3, Figure 4). Effet of UFD on plsm insulin level The serum insulin level signifintly deresed in STZ indued dieti ontrol group rts ws oserved. Three different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted group rts showed signifint (P <.1) inrese in the pnreti insulin ompred to dieti ontrol group rts on dy 28 (Tle 3, Figure 5). Effet of UFD on the level of glyted hemogloin The level of glyted hemogloin ws inresed in dieti group rts. Three different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted group rts, signifintly (P <.1) derese in the level of glyted hemogloin ompred to dieti ontrol groups rts on dy 28 ws oserved (Tle 3, Figure 6). Effet of UFD on the level of hexokinse The level of the hexokinse ws deresed in STZ indued dieti rts. Three different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted group rts, signifintly (P <.1) inresing the level of hexokinse ompred to dieti ontrol groups rts on dy 28 (Tle 3, Figure 7). The level of hexokinse t UFD doses 4 mg/kg ws mximum intensifition t ompred to other group reeived different doses of UFD nd glienlmide. Effet of UFD on the levels of gluose-6-phosphtse In the STZ indued dieti rts inresed the level of gluose-6-phosphte. Three different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted groups rts, signifintly (P <.1) deresed the level of gluose-6-phosphte ompred to dieti ontrol groups rts on dy 28 (Tle 3, Figure 8). An UFD dose 4 mg/kg ws more effetive dose s ompred Triglyeride (mg/dl) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 11 Effet of UFD on level of triglyeride t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P<.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

10 Kumr et l. SpringerPlus 213, 2:639 Pge 1 of 2 HDL Cholesterol ( mg/dl) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 12 Effet of UFD on level of HDL (High density lipoprotein) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). to the different doses of UFD nd glienlmide dministrtion groups. Effet of UFD on the levels of frutose-1-6-iphosphtse To evlute the effet of different doses of UFD on distressed hepti tivity, we dministered UFD to STZ indued dieti rts. The level of frutose-1-6-iphosphte ws rehed higher in dieti rts. The dministrtion of different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted groups rts signifintly (P <.1) delining the level of frutose-1-6-iphosphtse (Tle 3, Figure 9). Effet of UFD on the level of totl holesterol The level of holesterol ws inresed in the STZ indued dieti rts. The dministrtion of different doses of UFD signifintly deresed the level of totl holesterol (Tle 3, Figure 1). Effet of UFD on the levels of serum triglyerides It is evident from the figure tht the dministrtions of STZ to Wistr (lino strin) rts show n inrese in the serum triglyeride level. The dministrtion of different doses of UFD the level of serum triglyeride suordinte to good extent. The mximum lowering the serum triglyeride ws ppered in the group reeived UFD t dose (4 mg/kg) (Tle 3, Figure 11). Effet of UFD on the level of HDL holesterol It is preditle tht the level of HDL holesterol ws deresed in the STZ dieti rts. Upon the dministrtion of different doses of UFD, signifint inrese in the level of HDL holesterol s ompred to the dieti rts (Tle 3, Figure 12). Effet of UFD on the level of LDL holesterol It is evident from the Figure 13 tht STZ indued dieti rt showed n inrese in level of LDL holesterol. Tretment with different doses of UFD dereses the level of LDL holesterol. The figure suggests tht mximum derese in the higher level of LDL holesterol ws found in UFD (4 mg/kg) dose (Tle 3). LDL Cholesterol (mg/dl) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 13 Effet of UFD on level of LDL (Low density lipoprotein) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

11 Kumr et l. SpringerPlus 213, 2:639 Pge 11 of 2 VLDL Cholesterol ( mg/dl ) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 14 Effet of UFD on level of VLDL (Very low density lipoprotein) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). Effet of UFD on the level of VLDL holesterol STZ indued dieti rt lerly depited the inresed level of VLDL holesterol (Tle 3). Orl dministrtion of different doses of UFD nd glienlmide signifintly (P <.1) dereses the level of VLDL holesterol. The Figure 14 suggests tht UFD (4 mg/kg) dose ws more effetive in deresing the elevted level of VLDL holesterol. Effet of UFD on enzymti ntioxidnt mrkers Affet on enzymti ntioxidnt mrkers, the level of superoxidse dismutse (SOD), glutthione peroxidse (GPx) nd tlse (CAT) were inresed nd the level of mlondildehyde (MDA) ws signifintly deresed in STZ indued dieti groups rt. Tretment with the different doses of UFD (1, 2 nd 4 mg/kg) nd glienlmide (1 mg/kg) treted group rts signifintly (P <.1) inresed the level of SOD (Figure 15), GPx (Figure 16), CAT (Figure 17) nd deresed the level of MDA (Tle 4, Figure 18). Effet of UFD on liver histopthology Histopthology studies of the STZ indued dieti rt showed inresed level of ft umultion nd lrge re of heptoytes tken over y ft droplet (Figure 19). Orl dministrtion of UFD with different doses improved the kidney histopthology. UFD dose (1 mg/kg) showed deposition of ft s ompred to the norml rt, UFD dose 2 mg/kg histopthology showed few mro droplets of ft nd UFD dose 4 mg/kg shown no ft deposition s shown in the liver histopthology. Glienlmide treted group rt histopthology hd shown norml liver (Figure 2). Effet of UFD on kidney histopthology Histopthology of STZ indued dieti rt kidney shows inflmmtion in lood vessels, ft deposition, hnges in size of glomerulus nd inreses the thikness of owmn psules. Orl tretment with different doses of UFD nd glienlmide showed the hnges in STZ indued dieti groups rt. Different doses of UFD showing less ft deposition, norml size of glomerulus nd owmn SOD (U/mg of protein) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 15 Effet of UFD on level of SOD (Superoxide dismutse) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

12 Kumr et l. SpringerPlus 213, 2:639 Pge 12 of 2 CAT (U/mg of protein) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 16 Effet of UFD on level of CAT (Ctlse) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns). psules t dose dependent mnner. The UFD (4 mg/kg) dose showed norml histopthology of the kidney s ompred to the norml ontrol (Figures 21 nd 22). Effet of UFD on pnres histopthology 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 different doses of UFD nd glienlmide restored the neroti nd firoti hnges ndrisedthenumerofβ ells(figures23nd24). Effet of UFD on hert histopthology STZ indued dieti rt showed the inresed degree of interstitil spe nd distort interlted dis (Figure 25). STZ indued dieti rt, tretment with ltered doses of UFD nd glienlmide showed effet on the hert histopthology. STZ indued dieti rt treted with UFD (1 mg/kg) dose showed less interstitil spe nd distort interlted dis ompred to the dieti ontrol, other UFD (2 mg/kg) dose showing some interstitil spe nd UFD (4 mg/kg) dose showed the norml histopthology of hert like glienlmide treted group rt (Figure 26). Glienlmide treted group exhiited the histopthology like the norml ontrol. Disussion Aegle mrmelos Corre rih soure of mny ompounds. The methnoli extrt ws sujeted to olumn hromtogrphy nd isolted the ompound. The isolted ompound exhiited lue fluoresene nd UV sorption mxim t 256, 277 nd 332 nm nd IR sorption nd t 172 m -1 for δ-ltone ring suggested oumrin nture of the moleule. It lso hd IR sorption nds for hydroxyl groups (3452, 341, 3325 m -1 ) nd n romti ring (1629, 1515 m -1 ). On the sis of mss spetrum nd 13 C NMR spetr the moleulr ion pek of the ompound ws determined t m/z 486 onsistent to the moleulr formul of oumrin diglyoside C 21 H 26 O 13. The 1 H NMR spetrum showed the presene of two AB-type doulets t δ 6.83 (J = 9.2 Hz) nd 7.47 (J = 9.2 Hz) ssigned to vinyli H-3 nd H-4 protons, respetively. A GPx (U/mg protein) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg) Figure 17 Effet of UFD on level of GPx (Glutthione peroxidse) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

13 Kumr et l. SpringerPlus 213, 2:639 Pge 13 of 2 Tle 4 Effet UFD on ntioxidnt enzyme t end of the study S. No. Biohemil prmeter 1 SOD (U/mg of protein) 2 CAT (U/mg of protein) 3 GPx (nmole/mg of protein) 4 MDA (nmole/mg of protein) Norml ontrol Norml ontrol + UFG (4 mg/kg) STZ-dieti ontrol STZ dietes + UFD (1 mg/kg) STZ dietes + UFD (2 mg/kg) STZ dietes + UFD (4 mg/kg) STZ dietes + Glienlmide (1 mg/kg) 27.8 ± ± ± *** ± * ± ** ± *** ± *** ± ± ± *** 79.8 ± * 1.8 ± * ± 2.15 *** ±.861 *** 34.4 ± ± ±.77 *** 22.4 ±.59 * 26.4 ±.562 ** 32 ±.712 *** 29.6 ±.514 ***.212 ± ± ±.11 ***.431 ±.13 *.331 ±.12 **.251 ±.7 ***.294 ±.5 *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non signifint; ANOVA, followed y Dunnett s multiple omprison test. Compred to vehile ontrol. Compred to dieti ontrol. one-proton doule doulet t δ 7.55 (J = 9.8, 2.8 Hz) nd two one-proton doulets t δ 7.2 (J = 2.8 Hz) nd 6.4 Hz (J = 9.8 Hz) were sried to oumrin H-6, H-8 nd H-5 protons, respetively. Two one-proton doulets t δ 5.27 (J = 3.6 Hz) nd 4.99 (J = 3.6 Hz) were ounted to α-oriented nomeri H-1 I nd H-1 II protons, respetively. The other sugr protons resonted etween δ The 13 C NMR spetrum displyed signls for nine oumrin rons in the rnge of δ , nomeri rons t δ 13.8 (C-1 I ), (C-1 II ) nd other sugr rons etween δ The existene of NMR H-2 I signl in the deshielded region t δ 4.31 nd ron C-2 I signl t δ indited (2 I 1 II ) linkge of the sugr units. The HMBC spetrum of the oumrin showed intertions of H-6, H-8 nd H-1I with C-7; H-3 nd H-4 with C-2; nd H-2 I,H-2 II nd H-3 II with C-1 II.The 1 Hnd 13 CNMR spetrl dt of the oumrin nuleus were ompred with the reported dt of other oumrins (Ro et l. 29; Aslm et l. 212; Chkthong et l 212). On the sis of spetrl dt nlysis the struture of this ompound hs een eluidted s umelliferon-α-d-gluopyrnosyl- (2 I 1 II )-α-d-gluopyrnoside. Dietes (Type II) generlly ours due to humn genetilly suseptiility, s result loss of insulin produing pnreti β-ell ytotoxiity medited through the relese of nitri oxide (NO). Insulin dependent dietes mellitus (IDDM) is used y the progressive destrution of the insulin sereting pnreti β-ells. STZ is ytotoxi ompound otined from the soil miroes Streptomyes hromogenes. STZ minly penetrte the β-ells vi gluose trnsporter nd rek the DNA strnd in β-ells using the endogenous insulin relese (Kumr et l. 211). Due to rekge of DNA strnd leds to mendment of lood sugr level nd gluose onentrtions in lood. Severl plnt hve een ounted s n ntidieti effet y vriety of mehnisms suh s stimulting the regenertion of Islets of Lngerhns in the pnres, improving insulin sensitivity nd ugmenting gluose dependent insulin seretion in STZ indued dieti rts (Sezik et l. 25; Disy et l. 29). A lot of syntheti ntidieti drugs ville in the mrket ut sulfonylure suh s glienlmide often use s stndrd ntidieti drug in STZ indued dietes to ompre the effiy of vriety of ntihyperglyemi ompounds. (Kumr et l. 213). MDA (nmole/mg of protein) Norml Control Norml Control+UFD (4 mg/kg) Dieti Control UFD I (1 mg/kg) UFD II (2 mg/kg) UFD III (4 mg/kg) Glienlmide (1 mg/kg). Figure 18 Effet of UFD on level of MDA (Mlondildehyde) holesterol t different onentrtions on STZ indued dieti rts, ompred to stndrd drug Glienlmide; vlues re men ± SEM; n = 6; P <.5; P <.1; P <.1; P >.5 is onsidered s non-signifint (ns).

14 Kumr et l. SpringerPlus 213, 2:639 Pge 14 of 2 Figure 19 Effet of UFD on liver in different groups of rts: (A) Norml ontrol: Histopthology of norml ontrol group did not shown ny ft deposition nd other hnges (B) Dieti ontrol: Histopthology of dieti rt showed umultion of miro droplet of ft (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of the strting dose of tested drug shown some prt hving ft deposition whih ws less ompred to the dieti ontrol (yellow rrow). (D) UFD II (2 mg/kg): Histopthology of seond dose of tested drug showed very few miro droplet of ft (yellow rrow). (E) UFD III (4 mg/kg): Histopthology of other tested drug not showing ny ft deposition nd other hnges. (F) Glienlmide (1 mg/kg): Stndrd drug treted group shown histopthology similr to the norml ontrol groups. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 1. Aute toxiity studies of the iotive ompound of UFD reveled the non-toxi nture in the lower dose. There ws no lethlity or ny toxi retions found with the seleted doses of UFD until the end of the speifi study. The seletion of the doses ws done on the sis of lirtion urve (Slhuddin nd Jllpure 21). Orl gluose tolerne test ws performed for the identifition of the ltertion of rohydrte metolism during post gluose dministrtion. The different doses of the UFD signifintly ltered the lood gluose level s ompred to the gluose ontrol group rts. The result suggests tht the different doses of the UFD hve etter Figure 2 Effet of UFD on liver in different groups of rts: (A) norml ontrol: Norml ontrol not showing ny hnge in liver histopthology. (B) Dieti ontrol: Dieti ontrol rt shown ft deposition on the liver in the form of miro droplet. (yellow rrow) (C) UFD I (1 mg/kg): Tested drug some prt showed the deposition of miro droplet of ft (yellow rrow). (D) UFD II (2 mg/kg): Histopthology of tested drug showed some prt of miro droplet of ft deposition (yellow rrow). (E) UFD III (4 mg/kg): Histopthology of tested drug similr to the glienlmide treted drug. (F) Glienlmide (1 mg/kg): Stndrd drug treted group shown histopthology similr to the norml ontrol groups. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 4.

15 Kumr et l. SpringerPlus 213, 2:639 Pge 15 of 2 Figure 21 Effet of UFD photomirogrphs of histologil hnges in rt pnres: (A) Norml ontrol: norml histologil struture of rt pnres showed norml islet (white rrow) (B) Dieti ontrol: Histopthology of dieti ontrol rt showed fol nerosis (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug rt showed igger size of islet nd fol nerosis (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug rt showed fol nerosis (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug rt showed norml size of islet (white rrow) (F) Glienlmide (1 mg/kg): glienlmide treted rt pnres showed norml islet (white rrow). For eh group 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 1. gluose utiliztion pity. The possile mehnism of tion of the UFD my e due to insulin emission from the β-ell nd improved the gluose trnsporttion nd onsumption in the rts (Ceriello 25: Sntigu et l. 212). STZ indued dieti rt showed the inrese level of the lood gluose nd derese level of the plsm insulin. STZ destroy the β-ell in the pnres nd inrese the overprodution of gluose nd gluoneogenesis. Gluoneogenesis nd overprodution of the gluose is the prime ftor of the hyperglyemi (Ltner 1958). STZ indued dieti rts treted with the different doses of the UFD signifintly deresed the lood gluoselevelndimprovetheplsminsulinlevely regenertion of the β-ells. The possile mehnism of tion of the UFD my e stimulting the insulin seretion nd regenertion of the β-ells of the pnres Figure 22 Effet of UFD photomirogrphs of histologil hnges in rt pnres: (A) Norml ontrol: norml histologil struture of rt pnres showed norml islet (white rrow) (B) Dieti ontrol: Histopthology of dieti ontrol rt showed fol nerosis (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug rt showed igger size of islet nd fol nerosis (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug rt showed fol nerosis (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug rt showed norml size of islet (white rrow) (F) Glienlmide (1 mg/kg): glienlmide treted rt pnres showed norml islet (white rrow). For eh group 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 4.

16 Kumr et l. SpringerPlus 213, 2:639 Pge 16 of 2 Figure 23 Effet of UFD photomirogrphs of histologil on hert in different groups of rts: (A) Norml ontrol: Histopthology of norml ontrol group rt norml histopthology of hert (B) Dieti ontrol: Histopthology of dieti ontrol group rt showed inresed interstitil spe nd distort the interlted dis (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug shown deresed interstitil spe nd interlted dis (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug showed less interstitil spe (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug showed norml hert like the glienlmide (F) Glienlmide (1 mg/kg): Histopthology of glienlmide treted drug shown the norml histopthology of hert. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 1. or regenertion of the grnules in the β-ells nd enhned the ellulrity of the Islet of Lngerhns (Kumr et l. 213). The hypothesis further onfirmed y the pnres histopthology whih showed tht the UFD exhiit the protetive effet over the pnres ginst the miroil streptozotoin (Figures 23 nd 24). The UFD shows the similr mehnism of tion s glienlmide, stimulting the insulin seretion. The derese in ody weight ws found throughout the study in dieti ontrol group rts. The derese in Figure 24 Effet of UFD photomirogrphs of histologil on hert in different groups of rts: (A) Norml ontrol: Histopthology of norml ontrol group rt norml histopthology of hert (B) Dieti ontrol: Histopthology of dieti ontrol group rt showed inresed interstitil spe nd distort the interlted dis (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug showed deresed interstitil spe nd interlted dis (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug showed less interstitil spe (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug shown norml hert like the glienlmide (F) Glienlmide (1 mg/kg): Histopthology of glienlmide treted drug showed the norml histopthology of hert. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 4.

17 Kumr et l. SpringerPlus 213, 2:639 Pge 17 of 2 Figure 25 Effet of UFD on kidney in different groups of rt: (A) Norml ontrol: Norml ontrol histopthology showed norml size of glomerulus (green rrow) (B) Dieti ontrol: Histopthology of dieti ontrol rt showed inflmmtory ell in lood vessels (rown rrow) nd deposition of fts (yellow rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug showed inflmmtion in lood vessels (rown rrow) nd ft deposition (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug showed only ft deposition (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug showed norml kidney histopthology like the glienlmide group (F) Glienlmide (1 mg/kg): Glienlmide treted niml histopthology shown the norml kidney. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 1. ody weight due to gluoneogenesis, tolism of proteins nd fts. Ctolism whih is diretly ssoited with the hrteristi loss of ody weight due to inresed musle destrution or degrdtion of struturl proteins (Pulsen 1973; Shirwikr et l. 24; Shirwikr et l. 26). In this mnusript, results suggest tht STZ indued dieti groups rts treted with different doses of UFD signifintly inresed the ody weight s ompred to the dieti ontrol group rts in dose dependent mnner. The potentil mehnism of tion of the UFD showed Figure 26 Effet of UFD on kidney in different groups of rt: (A) Norml ontrol: Norml ontrol histopthology showed verge size of glomerulus (green rrow) (B) Dieti ontrol: Dieti ontrol histopthology showed ft deposition (yellow rrow) inflmmtory ell in lood vessels (rown rrow) (C) UFD I (1 mg/kg): Histopthology of tested drug showed inflmmtion in lood vessels (rown rrow) nd ft deposition (yellow rrow) (D) UFD II (2 mg/kg): Histopthology of tested drug showed ft deposition in few ple (yellow rrow) (E) UFD III (4 mg/kg): Histopthology of tested drug showed norml glomerulus ut slightly igger in size (F) Glienlmide (1 mg/kg): Glienlmide treted niml histopthology shown the norml kidney. The smples were otined from the sme liver ntomil regions. For eh group, 6 rts were exmined nd 8 pitures were tken. The ove piture for eh group ws hosen rndomly from the 8 pitures in this group. Originl mgnifition, 4.

18 Kumr et l. SpringerPlus 213, 2:639 Pge 18 of 2 the protetive effet ginst the ontrolling the musle wsting (reversl of gluoneogenesis). STZ indued dieti rts showed the lood gluose level inresed, inrese level of gluose, gluose dd to the RBC in N terminl of hemogloin hin nd produing the glyted hemogloin (H1) nd inresed the level of glyted hemogloin in STZ indued dieti rts. In norml, glyted hemogloin mke up % of totl hemogloin nd smll portion of lood gluose, usully etween 4.5-6%, is ovlently onded to the red lood ells in hemogloin (Kumr et l. 213), ut the level of glyted hemogloin ws inresed in dieti mellitus ptient due to n exess of gluose present in the lood rets with hemogloin to form glyted hemogloin. The level of glyted hemogloin ws inresed upto 16% in dietes mellitus ptients (Koeing et l. 1976). Glyted hemogloin n e used s n inditor of metlli ontrol of dietes sine glyohemogloin levels pproh norml vlue in dietes in metoli ontrol. In this investigtion the level of glyted hemogloin ws elevted more thn 4 times to the norml ontrol rts. Tretment with different doses of UFD signifintly rought k the inresed level ner norml levels (Tle 3), whih indite the improved level of glyemi ontrol. The possile mehnism of tion of the UFD in the glyted hemogloin my e deresing the lood gluose level nd inhiit the ddition of the gluose with the hemogloin. Hyperholesterolemi nd hypertriglyeridemi re mostly found in the dietes due to lipid normlities (Shepherd 25). These re the mjor ftor involved in rising of oronry hert disese nd theroslerosis, whih re the seondry omplition ompnying during dietes (Annthn et l. 23). The level of triglyeride inresed due to insulin defiieny resultnt filure to tivte lipoprotein lipse therey using hypertriglyeridemi (Shirwikr et l. 25). In dietes, the deposition of the holesterol in the peripherl tissue is rrying y LDL nd VLDL, peripherl tissue to survive nd then exretion of holesterol done y HDL. Hene inresed level of LDL nd VLDL is therogeni. The level of serum lipids ws elevted 2 times more s ompred to the norml ontrol rts. Tretment of different doses of UFD signifintly ontrols the inresed level of serum lipids (Triglyeride, Low density lipoprotein, VLDL) nd signifintly inresed the level of HDL in dieti ontrol rts. Ltely, mny investigtors hve een onentrted on the role of oxidtive stress in dietes. The investigtor lims tht oxidtive stress plys n importnt roleinthedevelopmentofthedietiomplitions (Sepii-Dinçel et l. 29). SOD, CAT, GPx plys signifint role in preventing the ell dmging from oxidtive stress. During the oxidtive stress, prodution of free rdil strts, one generted, it ontinuously ret to eh other nd formed the new free rdils (Kumr et l. 213). These free rdils ret with ll iologil sustnes (minly polyunsturted ftty ids) in the ody nd ontinuous retion of the free rdil led to lipid peroxidtion. Inresed level of lipid peroxidtion in the ody derese the memrne fluidity, hnge the memrne ound reeptor nd impired enzyme tivity of memrne funtion (Arulselvn nd Surmnin 27). In our investigtion, the level of SOD, CAT, GPx ws deresed nd the level of MDA (s n inditor of LPO) inresed in STZ indued dieti rts, hving high rte of free rdil genertion. But tretment with different doses of UFD signifintly deresed the level of MDA. The deresed in the level of MDA, n inrese in the level of GPx ws oserved, whih led to detivtion of LPO retion. ROS (Retive oxygen speies) diretly eliminted y primry enzyme suh s SOD nd CAT. SOD, is ple of hnging the superoxide rdil nions (O 2 - )intohydrogen peroxide (H 2 O 2 ) nd CAT is ple to the redution of hydrogen peroxide nd involved in detoxifition of hydrogen peroxide (H 2 O 2 ) onentrtion. Some time in dietes the level of SOD ws inresed without inresing the level of GPx, tht in the ell fing the overlod of peroxidses. Then the ell peroxide rets with the trnsitionl metls nd immeditely formed the hydroxyl rdils, prodution of hydroxyl rdils is very hrmful to the ells (Hlliwell nd Gutteridge 1989). STZ indued dietes intivte the tivted ntioxidnt enzyme suh s SOD, CAT, nd GPx y flututing these proteins thus produing indued oxidtive stress, ontinuously oxidtive stress used the LPO (Kennedy nd Lyons 1997). In our investigtion the SOD nd CAT signifintly deresed the dietes s result of non-enzymti glyosyltion nd oxidtion (Al-Azzwie nd Alhmdni 26). The possile mehnism of tion of the UFD my e enhning the level of the endogenous ntioxidnt enzymes. Liver is vitl orgn tht ply n importnt role in defense of the postprndil hyperglyemi nd involved in the gluose metolism (synthesis of glyogen). In liver, gluose is onverted into gluose-6-phosphtse y the help of hexokinse (Lth nd Pri 23; Bquer et l. 1998). STZ indued dieti rts derese glyolysis, distur the pity of the liver to synthesize glyogen nd deresed the level of hexokinse. Deresed level of hexokinse showed, n effet on glyolysis nd inhiits the utiliztion of gluose for energy prodution (Rju et l. 21). The STZ indued dieti rts treted with different doses of UFD rought k the tivity of this enzyme ner to norml ontrol nd inreses the utiliztion of gluose for energy onversion. Another liver vitl enzyme is gluose-6-phosphtse whih regultes the gluose metolizing enzyme. In STZ indued dieti rts inresed level of gluose-6-phosphtse oost the prodution of fts from rohydrtes nd inresed the fts deposition in the liver nd kidney (Liu et l. 1994). Some investigtors