Kumar et al. Kumar et al. BMC Complementary and Alternative Medicine 2013, 13:273

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1 Umelliferone β-d-glctopyrnoside from Aegle mrmelos (L.) corr. n ethnomedicinl plnt with ntidietic, ntihyperlipidemic nd ntioxidtive ctivity Kumr et l. Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273

2 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 RESEARCH ARTICLE Open Access Umelliferone β-d-glctopyrnoside from Aegle mrmelos (L.) corr. n ethnomedicinl plnt with ntidietic, ntihyperlipidemic nd ntioxidtive ctivity Viks Kumr 1*, Dnish Ahmed 1, Amit Verm 1, Firoz Anwr 2, Mohmmed Ali 3 nd Mohd Mujee 3* Astrct Bckground: Aegle mrmelos (L.) Corr. (Rutcee), commonly known s el, is used to tret fevers, domen pin, plpittion of the hert, urinry troules, melncholi, norexi, dyspepsi, dietes nd dirrhe in Indin trditionl systems of medicine. The oject of the present study ws to evlute the ntidietic, ntihyperlipidemic nd ntioxidnt oxidtive stress of umelliferone β-d-glctopyrnoside (UFG) from stem rk of Aegle mrmelos Corre. in STZ (streptozotocin) induced dietic rt. Methods: Dietes ws induced in rt y single intrperitonel injection of STZ (6 mg/kg). The rt ws divided into the following groups; I norml control, II dietic control, III UFG (1 mg/kg), IV UFG (2 mg/kg), V UFG (4 mg/kg), VI Glienclmide (1 mg/kg, p.o., once dily dose). Dietes ws mesured y chnge the level lood glucose, plsm insulin nd the oxidtive stress were ssessed in the liver y estimtion of the level of ntioxidnt mrkers i.e. superoxide dismutse (SOD), glutthione peroxidse (GPx), ctlse (CAT) nd Mlondildehyde (MDA) nd ntihyperlipidemic effect ws mesured y estimtion of totl cholesterol, triglycerides, LDL (low density lipoprotein) cholesterol, HDL (high density lipoprotein) cholesterol, VLDL (very low density lipoprotein) cholesterol. However in study, the incresed ody weight ws oserved nd utiliztion of glucose ws in the orl glucose tolernce test. Result: Dily orl dministrtion of different dose of UFG for 28 dys showed significntly (P <.1) decresed in fsting lood glucose level nd improve plsm insulin level s compred to the dietic control group. Also it significntly (P <.1) decresed the level of glycted hemogloin, glucose-6-phosphtse, fructose-1-6-iphosphte nd incresed the level of hexokinse. UFG tretment decresed liver MDA nd incresed the level of SOD, GPx nd CAT. UFG tretment of lipids it s incresed the level of cholesterol, triglycerides, VLDL, LDL cholesterol nd decresed the level of HDL cholesterol. Histologiclly, inflmmtory cell in lood vessels, interclted disc, ft degenertion nd focl necrosis oserved in dietic rt orgn ut ws less ovious in UFG treted groups. The mechnism of ction of UFG my e due to the incresed level of pncretic insulin secretion nd effect on the ntioxidnt mrker. Conclusion: UFG posses n ntidietic, ntioxidnt nd ntihyperlipidemic effect on the STZ induced dietic rt. Hence it could e the etter choice to cure the dietes. Keywords: Umelliferone β-d-glctopyrnoside, Streptozotocin, Antidietic, Antihyperlipidemic, Glienclmide * Correspondence: phviks@gmil.com; mohdmujee72@gmil.com 1 Deprtment of Phrmceuticl Sciences, Fculty of Helth Sciences, Sm Higginottom Institute of Agriculture, Technology & Sciences, Allhd, Uttr Prdesh 2117, Indi 3 Deprtment of Photochemistry & Phrmcognosy, Fculty of Phrmcy, Jmi Hmdrd, New Delhi 1162, Indi Full list of uthor informtion is ville t the end of the rticle 213 Kumr et l.; licensee BioMed Centrl Ltd. This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

3 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 2 of 19 Bckground Dietes mellitus (DM) hs very long historicl ccounts; it first points up in the medicl text of severl ncient cultures over 2 yer go. According to the reports tht, 135 million dults ffected from the dietes mellitus in yer 1995 worldwide nd the dt will increse to 3 million in the yer 225 [1,2]. Dietes mellitus is very common helth prolem rise worldwide rpidly, due chnging the food hit, lifestyle nd lrgely consumption of fst food. Mjor reson is genertion of free rdicl formtion, free rdicl genertion cused y degenertion of crohydrtes, lipid nd protein metolism y incresed lood glucose level (hyperglycemi) resulting from the defects in insulin secretion, insulin ction or oth. Elevted glucose production cuses oxidtive stress nd s result there is increse in mitochondril rective oxygen species (ROS), non-enzymtic glyction of proteins nd glucose utoxidtion [3]. In dietes, incresed oxidtive stress is due to genertion of free rdicl nd reduction of ntioxidnt defenses [4]. Endogenous ntioxidnt enzymes re responsile for the detoxifiction of injurious oxygen rdicls. Evidences from epidemiologicl nd iologicl studies hve estlished tht rective oxygen species (ROS) re involved in vriety of physiologicl nd pthologicl processes [5]. Different grdes of synthetic drugs, herl formultion ville in the mrket therefore they re investigted with renewed interest ll over the world [6,7]. A lot of clsses of synthetic drug re ville in the mrket ut quite few herl drugs re eing employed in the tretment of dietes mellitus. Only metformin is the one exmple of drug which is otined from the her (Gleg officinlis) with very long history of use for dietes. Still reserching is going on to find out the more effective herl drug to cure the dietes nd reduced the free rdicl formtion with minimized side effect. Aegle mrmelos Corre. (Rutcee) plnt is found in ll over Indi nd lso clled s IndinQuince, holy fruit (According to Hindu mythology it is holy plnt), Bengl quince, Golden Apple (English), ilvm (Tmil) Bilv, Sriphl, Shivdrum, Shivpl (Snskrit) Bil (Gujrti), Bel (Bngli) nd Bel (Hindi) [7-9]. Different prts of the plnt (fruit, seed, leves, root, rk nd flowers) re used in preprtion of vrious herl preprtions. The used of el ws hving very long history. The most commonly used prt is the fruit; fruit juice ws strined nd sweetened to mke drink similr to lemonde. In Ayurved fruit re used for hert, stomch, intestinl tonic, chronic constiption nd dysentery; some forms of indigestion, typhoid, deility, fever, hemorrhoids, hypochondri, melncholi nd for hert plpittion. Vrious chemicl constituents like Alkloids, coumrins nd steroids hve een isolted nd chrcterized from different prt of the tree, such s leves, fruit, wood, root nd rk [1]. The present reserch exertion ws tken up to evlute the nti-dietic ctivity of Umelliferone β-dglctopyrnoside isolted from the stem rk of Aegle mrmelos Corre. Since in the previous reserch [11] it ws estlished tht Umelliferone is potent free rdicl scvenger nd works s ntioxidnt. Till dte no study hs een reported on the ntioxidnt ctivity of Umelliferone β-d-glctopyrnoside nd the mjor root cuse of dietes mellitus is the development of free rdicls which destroys the β-cells of the pncretic islets [3], responsile for the secretion of insulin. Therefore, we hve tken up the isolted compound for the evlution ginst the dietes, hyperlipidemi nd oxidtion. Methods Generl Melting point ws set up on Veego, Model No. MPI is melting point pprtus nd re uncorrected. 1 HNMR spectr were recorded on Bruker Avnce II 4 NMR Spectrophotometer nd 13 C NMR spectr on BrukerAvnce II 1 NMR Spectrophotometer in DMSO using TMS s internl stndrd. Mss spectr were otined on the VG- AUTOSPEC spectrometer. UV λmx (DMSO) were recorded on Shimdzu UV-17 nd FT-IR (in 2. cm-1, flt, smooth, Aex) were tken on Perkin Elmer Spectrum RX-I spectrophotometer. Mteril The stem rk of Aegle mrmelos Corre. collected from the otnicl grden, Deprtment of Phrmceuticl Sciences, Fculty of Helth Sciences, Sm Higginottom Institute of Agriculture, Technology & Sciences Deemed University nd uthenticted y Dr. Imrn Kjmi (Phrmcognosist) nd specimen voucher (SIP/HD/54/ 12) of the plnt smple respectively hve een deposited in the herrium of Siddhrth Institute of Phrmcy, Dehrdun, Uttrkhnd, Indi. Chemicl Silic gel (6 12 mesh) (Nichols Indi Pvt. Ltd) nd glss column were used for column chromtogrphy. Streptozotocin (Sigm Chemicl Co. USA), GOD/POD kit, Cholesterol kit, Triglyceride kit, (Spn, Indi), Glienclmide (purity > 99%), Croxyl methyl cellulose (SD fine, Indi), chemicls nd other solvents used for the chromtogrphy isoltion nd experimentl protocol of nlyticl grde nd were purchsed from respective vendor, Allhd, Indi. Extrction nd isoltion The shde dry stem rk of Aegle mrmelos Corre (2 kg) ws extrcted with methnol (5 L) t the 45 C for 72 h [12,13]. After extrction totl filtrte ws concentrted to dryness in rottory vcuum evportor t 4 C

4 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 3 of 19 to otin slurry (322 gm). The slurry ws dissolved in smll mount of methnol nd ws sored on silic gel (6 12 mesh). It is sujected to silic gel column 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 ech grdient system), yielding 22 frctions collected frction spotted on pre coted silic gel TLC plte nd the frction hving the sme R f vlue pooled together in 7 frctions. Frctions 2 4 (13.5 g) were comined seprted on silic gel column (CHCl 3 /MeOH, 3:1), nd rechromtogrphed on silic gel column (CHCl 3 /MeOH, 6:1 to 3:1), yielding 7 sutrctions. Compound 1 ws seprted first y norml phse silic gel column (CHCl 3 /MeOH, 3:1). Animls Swiss lino wistr rts (15 22 g) ws used for the study. The nimls were housed under stndrd conditions of temperture (25 ± 1 C), reltive humidity (55 ± 1%), 12 hr/ 12 hour light/drk cycles nd nimls were received stndrdpelletdiet(liptonrtfeed,ltd.,pune)withndwter d liitum. The experimentl protocol ws pproved y the Institutionl Animl Ethicl Committee of Siddhrth Institute of Phrmcy (1435/PO//11/CPCSEA). Acute toxicity study The toxicity study ws conducted s per the guidelines of CPCSEA, rticle no 42. A seprte experiment performed for determintion of ny toxic effect of the test drug. For cute toxicity study, norml helthy wistr rts were fsted overnight (16 hour) nd rndomly divided into different groups nd ech groups contin rts (n = 1). Wistr rt ws treted with strting doses (.5,.1,.5 nd.1 g/kg ody weight) of test compound nd the control group ws treted with vehicle lone (CMC 2%; 1 ml/kg ody weight). All the niml groups llowed for food nd wter d liitum nd were oserved over period of 2 h for chnging in vrious utonomicl (defection nd urintion), neurologicl (touch, rectivity, spontneous, pin response nd git) nd ehvior (lertness, restlessness, irritility, nd ferfulness) responses nd fter 24 nd 48 h for mortlity [14,15]. If mortlity cused y the compound within this period of the time ws oserved [16]. Assessment of UFG in orl glucose tolernce test Assessment of orl glucose tolernce test, helthy rts were divided into seven groups of six nimls ech [17]. Group I. Norml control rts received CMC. Group II. Norml control rts received UFG (4 mg/kg p.o.). Group III. Glucose (2 gm/kg) received rts. Group IV. Glucose treted dietic rts received UFG (1 mg/kg p.o.). Group V. Glucose treted dietic rts received UFG (2 mg/kg p.o.). Group VI. Glucose treted dietic rts received UFG (4 mg/kg p.o.). Group VII. Glucose treted dietic rts received glienclmide (1 mg/kg p.o.). All group nimls received drug nd vehicle orlly. All the nimls were received glucose (2 g/kg) 3 min fter dosing. The lood smple ws collected from puncture of retro-oritl of n eye; their glucose tolernce ws studied up to 2 h t regulr intervl of, 3, 6, 12 min ech. Induction of dietes Wistr rts were injected dietes y single intrperitonel injection of streptozotocin (6 mg/kg ody weight). Volume of STZ 1 ml/kg ody weight prepred y STZ dissolving in freshly prepred.1 M citrte uffer (ph = 4. 5) [18]. After 3 dys dministrtion of STZ (streptozotocin) lood glucose level of rts were estimted. Rts with lood glucose level of 22 mg/dl eyond considered s dietic [19]. Experimentl design Wistr rts were divided into seven groups nd six nimls in ech group [2]. Group I. Norml control rts received citrte uffer (ph = 4.5) for 28 dys (1 ml/kg p.o.). Group II. Norml control rts received UFG (4 mg/kg p.o.) nd continued for 28 dys. Group III. STZ-dietic rts received vehicle only. Group IV. STZ treted dietic rts received UFG (1 mg/kg p.o.) nd continued for 28 dys. Group V. STZ treted dietic rts received UFG (2 mg/kg p.o.) nd continued for 28 dys. Group VI. STZ treted dietic rts received UFG (4 mg/kg p.o.) nd continued for 28 dys. Group VII. STZ treted dietic rts received glienclmide (1 mg/kg p.o.) nd continued for 28 dys. All groups niml received drug nd vehicle orlly, once dily. Blood ws collected on the regulr intervl y retro-oritl puncture under mild n nesthesi nd mesure lood glucose level nd collected lood smple ws centrifuged nd exmined for plsm glucose nlysis y GOD - POD method using the Glucose Estimtion Kit (Spn Dignostic, Indi). Biochemicl nlysis After 28 dys of tretment, lood smple ws drwn from puncture the retro oritl under mild nesthesi condition, collected lood ws centrifuged nd exmined for plsm glucose nlysis y GOD - POD method using the Glucose Estimtion Kit (Spn Dignostic, Indi). Other serum estimtion ws done spectrophotometriclly using stndrd kits which include serum

5 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 4 of 19 insulin (Spn Dignostic, Indi), totl cholesterol, HDL (High density lipoprotein) cholesterol (Spn Dignostic, Indi) nd triglyceride (Spn Dignostic, Indi). Hexokinse, glucose-6-phosphte nd fructose-1-6-iphosphtse ws estimted y the reported method of Brndstrup nd co-resercher [21]. Estimtion of ntioxidnt enzymes For estimtion of ntioxidnt enzymes, ll group rts liver tissue ws homogenized, centrifuged nd exmined for superoxide dismutse, ctlse, glutthione peroxidse, mlonldehyde levels ccording to the reported methods with minor modifiction [22-25]. Histopthology At 28 dy ll the niml scrificed under mild nesthesi nd different orgn (hert, liver, pncres nd kidney) of the niml ws isolted for histopthology studies. The isolted orgn (hert, liver, pncres nd kidney) tissue fixed with 4% neutrl uffered formlin, dehydrted y pssing through grded series of lcohol, emedded in prffin locks nd then 5 mm sections were developed using semi-utomted rottory microtome. Hemtoxylin nd eosin stin were used. Sttisticl dt nlysis All the dt were expressed s the men ± S. E. M. n nlysis of vrince (ANOVA) ws used for the sttisticl nlysis using Grph Pd Prism version 5.. The vlues were considered to e significnt when the P vlue ws.1. Result Chrcteriztion of isolted compound The methnolic extrct of stem of A. mrmelos ws sujected to column chromtogrphy. Frctions 4 6 were further purified y silic gel recolumn chromtogrphy nd the chromtogrphy purified of these frctions led to the isoltion of compound (5 mg). Isolted compound otined s yellowish coloured solid compound (3 gm), mp 24 C, R f :.35, (C 7 H 8 /(C 2 H 5 ) 2 O, 1:1), compound exhiited UV sorption nds t 33 nm (log ε 3.1), indicting coumrin derivtive. ESI- MS t m/z (rel. int.): 324 [M] + C 15 H 16 O 8 (1.8), 1 H NMR (DMSO-d 6 ): 7.9 (1H, dd, J 9.6, 2.8 Hz, H 6), 7.51 (1H, d, J = 9.1 Hz, H - 4), 7.2 (1H, d, J = 2.8 Hz, H - 8), 6.91 (1H, d, J = 9.1 Hz, H - 3), 6.91 (1H, d, J = 9.6 Hz, H-5),5.12(1H,d 1,J=7.2Hz,H-1 1 ), 4.36 (1H, H ), 3.82 (1H, H -2 1 ), 3.78 (1H, H -3 1 ), 3.67 (1H, H -4 1 ), 3.16 (2H, H -6 1 ). 13 C NMR (DMSO-d 6 ): ( C-2), (C-3), (C-4), (C-5), (C-6), (C- 7), 13.25(C-8), (C-9), (C-1), (C-1 I ), (C-2 I ), (C-3 I ), (C-4 I ), (C-5 I ), 62.5 (C-6 I )(Tle1).IRγ mx (KBr): 3435, 339, 2936, 2851, Tle 1 1 H nd 13 C NMR vlues of Umelliferone β-dglctopyrnoside Position 1 H 13 C Coupling constnts in Hertz re provided in prenthesis. 172, 167, 1515, 1458, 1425, 1337, 1278, 1224, 1115, 171 cm -1 (Figure 1) (Additionl File 1: Spectrl Dt of umelliferone β-d-glctopyrnoside). Acute toxicity study An cute toxicity study reveled the non-toxic nture of the UFG. There ws no lethlity or ny toxic rections found t ny of the doses selected until the conclusion of the study period. Effect of UFG on orl glucose tolernce test The lood glucose level in rt fed on norml diet (norml control, group I) ws lmost constnt throughout the complete study. The norml control group rt received UFG (4 mg/kg) dose significntly showing the etter utiliztion of glucose (Group II) s compred to Figure 1 Structure of umelliferone β-d-glctopyrnoside.

6 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 5 of 19 Figure 2 Effect of umelliferone β-d-glctopyrnoside on fsting plsm glucose on orl glucose tolernce test t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P<.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). the norml control group rt. Glucose induced higher lood glucose level group rt treted with different doses of UFG inhiit the high lood glucose level. Three different doses of UFG significntly (p <.1) inhiit the high lood glucose level y 13.9%, 17.6% nd 29.8% t the tested doses of 1 mg/kg, 2 mg/kg nd 4 mg/kg respectively s shown in Figure 2. Glienclmide (1 mg/kg) significntly (p <.1) inhiit glucose excursion y 28% (Tle 2). Effect of UFG on lood glucose level The lood glucose level of the rt fed norml diet (norml control) remin unchnged t throughout the experimentl study (Group I). In the other group received norml fed nd dose UFG 4 mg/kg (Group II) shown the lood glucose level ner the norml control group rt. On the contrry, the lood glucose level of STZ induced dietic rts ws incresed significntly (Group III). STZ dietic rt treted with different doses of UFG shown significntly (P <.1) lowering the lood glucose level t dose dependent mnner shown in Tle 3. On the other hnd, glienclmide (1 mg/kg) significntly inhiit the lood sugr level (Figure 3). Effect of UFG of plsm insulin The levels of plsm insulin in dietic groups were significntly decresed s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). Different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) received groups significntly (p <.1) increse the level of plsm insulin. The result suggests tht the plsm insulin level of UFG 4 mg/kg mximum increse when compre with the 1 mg/kg, 2 mg/kg nd glienclmide (Figure 4). Effect of UFG on totl cholesterol To evlute the effect of UFG on totl cholesterol level, the dietic rt received different doses (1 mg/kg, 2 mg/kg nd 4 mg/kg) of UFG nd glienclmide Tle 2 Effect of umelliferone β-d-glctopyrnoside on lood glucose levels in orl glucose tolernce test in normoglycemic rts S. No. Tretment Dose Men lood glucose concentrtion ± SEM (mg/dl) Time min Time 3 min Time 6 min Time 12 min 1 Norml Control ± ± ± ± Norml Control + UFG 4 mg/kg 86.8 ± ± ± ± Glucose Control ± ± ± ± UFG I 1 mg/kg 88.4 ± ± ns ± 1.21 * 16 ± *** 5 UFG II 2 mg/kg 87.4 ± ± * ± ** 11.4 ± 1.32 *** 6 UFG III 4 mg/kg 88.8 ± ± ** 19 ±.732 *** 87 ± *** 7 Glienclmide 1 mg/kg 89 ± ± 1.68 ** ± 1.34 *** 88.6 ± *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non significnt; ANOVA, followed y Dunnett s multiple comprison test.

7 Tle 3 Effect of umelliferone β-d-glctopyrnoside on iochemicl prmeters in STZ-induced dietic rts S. No. Biochemicl prmeter Norml control Norml control + UFG (4 mg/kg) STZ-dietic control STZ dietes + UFG STZ dietes + UFG STZ dietes + UFG STZ dietes + (1 mg/kg) (2 mg/kg) (4 mg/kg) glienclmide 1 Fsting plsm glucose (mg/dl) 91.2 ± ± ± *** ± ** ± *** ± *** ± 1.99 *** 2 Fsting Plsm Insulin (μu/ml) 11.2 ± ± ±.383 *** 4.4 ±.59 * 6.8 ±.374 ** 9.4 ±.519 *** 8.6 ±.69 *** 3 Glycted Hemogloin (A1c) (%) 1.4 ± ± ± 2.49 *** 3.8 ±.184 * 3.42 ±.182 ** 1.86 ±.161 *** 2.4 ±.212 *** 4 Hexokinse (μg/mg of tissue) 15.4 ± ± ± *** ± ** ± ** ± *** 139 ± *** 5 Glucose-6-Phosphtse (unit/mg of tissue) 6 Fructose-1-6-iphosphtse (unit/mg of tissue) 1 ± ± ±.59 *** 14.4 ±.612 ns 12.8 ±.374 * 1.8 ±.583 *** 11.6 ±.244 *** 3.8 ± ± ± *** 47.4 ± * 38.2 ± ** 32.2 ±.861 *** 34.8 ±.583 *** 7 Totl Cholesterol (mg/dl) 66.6 ± ± ± *** 97.2 ± ** 82.2 ± ** 61.2 ± *** 66.2 ± *** 8 Triglycerides (mg/dl) 82.4 ± ± ± *** ± * ± 1.66 ** 96.2 ± *** 18.4 ± 1.71 *** 9 Totl HDL Cholesterol (mg/dl) 58.6 ± ± ± *** 42.8 ± * 5.4 ±.927 ** 59.2 ± 1.68 *** 55.8 ± *** 1 Totl LDL Cholesterol (mg/dl) 37.9 ± ± ± *** ± 1.21 ** 65.5 ± *** 44.4 ± *** 61.8 ± *** 11 Totl VLDL Cholesterol (mg/dl) ± ± ±.647 *** ±.232 * ±. 331 ** ±.279 *** ±.215 *** 12 Weight Vrition (g) ± 196 ± ± ± *** 19.4 ± *** 195 ± *** 23.2 ± *** ± *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non significnt; ANOVA, followed y Dunnett s multiple comprison test. Compred to vehicle control. Compred to dietic control. Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 6 of 19

8 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 7 of 19 Fsting plsm glucose (mg/dl) Figure 3 Effect of umelliferone β-d-glctopyrnoside on fsting plsm glucose t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). (1 mg/kg). The level of cholesterol increses in dietic rt s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). Different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) received group decresing the level of totl cholesterol s compred to the dietic control groups rt. UFG dose 4 mg/kg is more effective to decrese the level of totl cholesterol s compred to other group treted with different doses of UFG (1 mg/kg, 2 mg/kg) nd glienclmide (Figure 5). Effect of UFG on triglyceride The level of triglyceride is incresed in STZ induced dietic rt s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). Increse the level of triglyceride in STZ induced dietic group, treted with different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) significntly inhiit the level of triglyceride. UFG 4 mg/kg is most effective in dose on inhiiting the mximum level of triglyceride s compred to 1 mg/kg, 2 mg/kg doses of UFG nd glienclmide (Figure 6). Effect of UFG on totl HDL (high density lipoprotein) cholesterol To evlute the effect of UFG on totl HDL cholesterol, the level of HDL cholesterol ws decresed in STZ treted dietic rt s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). The effect of different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) significntly increses the level of HDL cholesterol. UFG 4 mg/kg doses shown the mximum incresing the level of HDL cholesterol s compred to 1 mg/kg, 2 mg/kg doses of UFG nd glienclmide (1 mg/kg) treted group (Figure 7). Effect of UFG on totl LDL (low density lipoprotein) cholesterol In STZ induced dietic rt incresed the level of LDL cholesterol s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt. The effect of different doses of UFG nd glienclmide in STZ induced dietic rt significntly (P <.1) inhiit the incresed level of LDL cholesterol (Tle 3). The 15 Insulin Level (µ U/mL) 1 5 c Dys Figure 4 Effect of umelliferone β-d-glctopyrnoside on level of plsm insulin t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

9 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 8 of 19 Totl Cholesterol ( mg/dl) Figure 5 Effect of umelliferone β-d-glctopyrnoside on level of totl cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). mximum decresing the LDL cholesterol level ws ppered in the group received UFG 4 mg/kg (Figure 8). Effect of UFG on totl VLDL (very low density lipoprotein) cholesterol To evlute the effect of UFG on VLDL (very low density lipoprotein) cholesterol, in STZ induced dietic rt incresed the level of VLDL cholesterol s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt. Dietic rt treted with different doses of UFG nd glienclmide significntly (P <.1) decresing the level of VLDL cholesterol. Dietic rts treted with UFG 4 mg/kg showed the mximum ugmenttion in the level of VLDL cholesterol s compred to other groups received different doses of UFG nd glienclmide (Figure 9). Effect of UFG on hexokinse The level of hexokinse decrese in STZ treted group s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). Dietic groups rt treted with different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) significntly increse the level of hexokinse. Dietic rt treted with dose 4 mg/kg UFG show mximum growth in the level of hexokinse s compred to other groups received 1 mg/kg, 2 mg/kg dose of UFG nd glienclmide (Figure 1). Effect of UFG on glucose-6-phosphte To evlute the effect of different doses of UFG on glucose-6-phosphte on dietic rt (Tle 3). The level of glucose-6-phosphte ws significntly incresed in dietic groups rt when compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt. Dietic rt treted with different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) significntly decrese the level of glucose-6-phosphte. UFG 4 mg/kg dose shown mximum increse in the level of glucose-6-phosphte when compred to 1 mg/kg, 2 mg/kg dose of UFG nd glienclmide (1 mg/kg) dose received groups (Figure 11). Effect of UFG on fructose-1-6-iphosphtse The orl dministrtion of different doses of UFG decreses the level of fructose-1-6-iphosphtse s compred to the norml control group nd UFG dose 4 mg/kg treted 2 Triglyceride (mg/dl) c Figure 6 Effect of umelliferone β-d-glctopyrnoside on level of triglyceride t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

10 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 9 of 19 HDL Cholesterol ( mg/dl) c Figure 7 Effect of umelliferone β-d-glctopyrnoside on level of HDL (High density lipoprotein) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). norml control group rt. The level of fructose-1-6- iphosphtse increse in STZ induced dietes (Tle 3). Dietic groups received different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) decrese the level of fructose-1-6-iphosphtse. The UFG dose 4 mg/kg shown the supreme diminish levels of fructose-1-6-iphosphtse comprison to other dietic treted group received dose 1 mg/kg, 2 mg/kg dose of UFG nd glienclmide (Figure 12). Effect of UFG on glycted hemogloin (A1c) The level of glycted hemogloin (A1c) incresed in STZ-induced treted dietic rts s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 3). The treted group with different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) nd glienclmide (1 mg/kg) significntly inhiit the level of glycted hemogloin (A1c). UFG dose 4 mg/kg treted group significntly inhiits the level of glycted hemogloin (A1c) s compred to 1 mg/kg, 2 mg/kg nd glienclmide (1 mg/kg) dose treted groups (Figure 13). Effect of UFG on mlondildehyde (MDA) The level of MDA in untreted dietic control rts ws significntly higher thn those in the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 4). Different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) dministered groups significntly (p <.1) inhiit the level of MDA. The mximum inhiition t the level of MDA seen in the group treted with 4 mg/kg of UFG. On the other hnd stndrd drug (glienclmide) lso inhiits the incresing level of MDA in dietic rts. The outcome suggests tht UFG dose 4 mg/kg is more effective thn the other doses of UFG nd glienclmide (Figure 14). Effect of UFG on glutthione peroxidse (GPx) The level of GPx ws significntly (p <.1) decresed in dietic control groups s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 4). Glienclmide (1 mg/kg) nd different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) received groups significntly (p <.1) increse the level of GPx. The outcome suggests tht LDL Cholesterol (mg/dl) Figure 8 Effect of umelliferone β-d-glctopyrnoside on level of LDL (Low density lipoprotein) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

11 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 1 of 19 VLDL Cholesterol ( mg/dl ) c Figure 9 Effect of umelliferone β-d-glctopyrnoside on level of VLDL (very low density lipoprotein) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). group which received UFG 4 mg/kg dose is increse the level of GPx s compred with other group rts received different doses of UFG nd glienclmide (Figure 15). Effect of UFG on superoxide dismutse (SOD) The level of ntioxidnt enzyme SOD ws significntly decresed in dietic control groups s compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 4). Glienclmide (1 mg/kg) nd different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) received groups significntly (p <.1) increse the level of SOD. The outcome suggests tht glienclmide nd ll the doses of UFG increse the level of SOD, ut UFG doses 4 mg/kg ws more effective in increse the level of SOD in dietic rt s compred with different doses of UFG nd glienclmide (Figure 16). Effect of UFG on ctlse (CAT) The level of CAT were significntly (p <.1) decresed in dietic control groups when compred to the norml control group nd UFG dose 4 mg/kg treted norml control group rt (Tle 4). Glienclmide (1 mg/kg) nd different doses of UFG (1 mg/kg, 2 mg/kg nd 4 mg/kg) received groups rt significntly (p <.1) increse the level of CAT. The dt suggest tht UFG 4 mg/kg dose ws more effective to increse the level of CAT in dietic rts s compred with other groups rt received different doses nd glienclmide (Figure 17). Chnges in ody weight At the end of 28 dys tretment, the ody weight of norml rts, UFG dose 4 mg/kg treted norml rt, dietic control, different doses of UFG nd glienclmide treted rts oserved (Tle 3). Dietic control group continued to lessen the weight till the conclusion of the study. Glienclmide nd UFG different doses (1 mg/kg, 2 mg/kg nd 4 mg/kg) treted rts significntly incresed the weight s compred to the dietic control rts (Figure 18). Effect of UFG on liver Histopthology studies of STZ induced dietic rt shown the ccumultion of ft nd lrge re of heptocytes tken over y mcro droplet of ft in the liver. Orl dministrtion of different doses of UFG improved the histopthology conditions. UFG dose 1 mg/kg dose Hexokinse (µg/mg of tissue) Figure 1 Effect of umelliferone β-d-glctopyrnoside on level of Hexokinse t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

12 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 11 of 19 Glucose-6-Phosphtse (Unit/mg of tissue) ns c Figure 11 Effect of umelliferone β-d-glctopyrnoside on level of Glucose-6-phosphtse t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). shown micro droplet of ft ccumultion, nother dose UFG 2 mg/kg dose shown some micro droplet of ft ccumultion on rt histopthology. Dose UFG 4 mg/kg shown the rt liver histopthology similr to the glienclmide drugs (Figures 19 nd 2). Effect of UFG on kidney Study of STZ induced dietes rt kidney histopthology shown inflmmtion in lood vessels, ft deposition, increse in the thickness of owmn cpsules nd chnge in size of the glomerulus. Tretment with different doses of UFG improves the injured rt kidney with incresing doses. The tretment with UFG 1 mg/kg dose showed improved kidney histopthology less inflmmtory lood vessels, less ft deposition s compred to dietic control. Tretment with UFG 2 mg/kg dose shown only ft deposition no inflmed lood vessels nd the dose UFG 4 mg/kg shown the norml histopthology there is no inflmmtory vessels nd no ft deposition (Figures 21 nd 22). Effect of UFG on pncres Histopthology studies of pncres of STZ induced dietic rt displyed reduction of the islets of lengerhens, dmged or reduced the size of β cells nd extensive necrosis chnges followed y firosis nd trophy. STZ induced dietic rt treted with different doses of UFG nd glienclmide restored the necrotic nd firotic chnges nd rised the numer of β cells (Figures 23 nd 24). Effect of UFG on hert Hert histopthology study of STZ induced dietic rt shown incresed the interstitil spce, interclted disc nd level of ft deposition. Orl dministrtion of UFG decresed the interstitil, interclted disc nd ft deposition t dose dependent mnner. UFG dose 4 mg/kg ws more effective to show norml histopthology of hert (Figures 25 nd 26). Discussion The isolted compound ws identified s umelliferone β- D-glctopyrnoside using different spectroscopy FT-IR, ESI-MS, 1 H-NMR, 13 C-NMR. IR sorption spectrum t 172 cm -1 nd the compound exhiit lue fluorescence nd UV sorption mxim t 256, 277 nd 33 NM for δ-lctone ring suggested coumrin nture of the isolted Fructose 1-6-iphosphte (unit/mg of tissue) c Figure 12 Effect of umelliferone β-d-glctopyrnoside on level of Fructose1-6-iphosphte t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

13 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 12 of 19 Glycerided Hemogloin (A1c) (%) c Figure 13 Effect of umelliferone β-d-glctopyrnoside on level of glycted hemogloin (A1c)(%) t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). compound. On the sic of 13 C NMR nd mss spectrum ESI-MS t m/z (rel. int.): 324 [M] + consistent with the moleculr formul of C 15 H 16 O 8. It lso hd IR sorption nds for hydroxyl groups (3435, 339, 2936 cm -1 ), nd n romtic ring (167, 1515 cm -1 ). The 1 H NMR spectrum showed the presence of two AB-type doule t δ 6.91 (J = 9. 1 Hz) nd 7.51 (J = 9. 6 Hz) ssigned to vinylic H-3 nd H-4 protons, respectively. One-proton doule douplet t δ 7.9 (J = 7.2, 2.8 Hz) nd two on proton doulets t δ 7.2 (J = 2.8 Hz) nd 6.91 Hz (J = 9.1) ws scried to coumrin H-6, H-8 nd H-5 protons, respectively. One-proton doulets t δ 5.12 (J = 7.2 Hz) were ccounted to α-oriented nomeric H-1 I protons, respectively. The other sugr protons resonted etween δ The 13 C NMR spectrum displyed signls for nine coumrin crons in the rnge of δ , nomeric cron t δ (C-1 I ) nd other sugr crons etween δ The existence of n NMR H-2I signl in the deshielded region t δ 3.82 nd cron C-2 I signl t δ indicted (2 I 1 II ) linkge of the sugr units. The HMBC spectrum of the coumrin showed interctions of H-6, H-8 nd H-1 I with C-7; H-3 nd H-4 with C-2; nd H-2 I. The 1 H nd 13 C NMR spectrl dt of the coumrin nucleus were compred with the reported dt of other coumrins [26-28]. On the sis of spectrl dt nlysis the structure of this new compound hs een elucidted s Umelliferone β-dglctopyrnoside. More thn 1 compounds lredy isolted from the Aegle mrmelos Corre ut umelliferone β-d-glctopyrnoside isolted first time in this plnt. Different prt of the plnt Aegle mrmelos hving the very long history to cure the dietes ut the lck of single ioctive compound ws still unknown. In this mnuscript we hve isolted the ioctive compound UFG (umelliferone β-d-glctopyrnoside) from the rk nd look into the ntidietic ctivity in norml nd dietic rt models. The result showed tht the different doses of UFG significntly decrese the lood sugr level, totl cholesterol, totl triglyceride, totl HDL, LDL cholesterol of dietes rts nd demonstrted the ntioxidnt ctivity (SOD, CAT, GPx, MDA) s compred to dietic control group nd glienclmide group rts. STZ is nitrosoure compound, widely used cytotoxic gent nd otined from the soil microe Streptomyces chromogenes, which effect on pncretic β-cells induced the dietes [29]. STZ ffects pncretic β-cells, secreted Tle 4 Effect on ntioxidnt enzyme t end of the study S. No. Biochemicl 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 control Norml control + UFG (4 mg/kg) STZ-dietic STZ dietes + STZ dietes + STZ dietes + STZ dietes + control UFD (1 mg/kg) UFD (2 mg/kg) UFD (4 mg/kg) glienclmide ± ± ± 4.5 *** ± ** ± ** ± *** 22.6 ± *** ± ± ± *** 85 ± * 97.8 ± ** 13.2 ± *** ± *** 33.2 ± ± ±.836 *** 21 ±.77 ** 24.6 ±.872 ** 31.2 ± 1.68 *** 28.8 ±.811 ***.241 ± ± ±.16 ***.396 ±.17 *.312 ±.8 **.274 ±.11 ***.261 ±.14 *** All vlues represent men ± SEM *P<.5; **P<.1; ***P<.1, ns < non significnt; ANOVA, followed y Dunnett s multiple comprison test. Compred to vehicle control. Compred to dietic control.

14 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 13 of 19 MDA (nmole/mg of protein) c. Figure 14 Effect of umelliferone β-d-glctopyrnoside on level of MDA (Mlondildehyde) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). on endogenous insulin which rrest stting the secretion of insulin; s result the level of insulin increse in lood, due to incresing levels of insulin it increses the level of lood glucose [3]. The Wister rts treted with different doses of UFG orlly exhiited norml ehviour till doses 1 mg/kg, these groups ehve normlly on touching nd pin response. There ws no lethlity or ny toxic rections found with the selected dose until the conclusion of the field. The dose of the test drug hs een selected on the sis of dose clirtion curve methods. Different ctegory of synthetic orl hypoglycemic gents currently ville in the tretment nd control of NIDDM (non insulin dependent dietes mellitus) including thizolidinediones, sulphonylures, igunides, α glucosidse inhiitors. Glienclmide (used s the reference hypoglycemic gent in this study) [31]. Glienclmide is sulphonylure clss of drug nd the most prole mechnism of ction is inducing insulin secretion. OGTT in the wistr rt model showed tht different doses of UFG significntly reduced the glucose excursion in dose dependent mnner. Norml control group unchnged t the end of the study ut norml control treted with UFG (4 mg/kg) dose treted group shown etter utiliztion of glucose s compred to the norml control. Different doses of UFG exhiited remrkle decresing the lood sugr lowering effect in the glucose tolernce test. UFG dose 4 mg/kg is more effective thn the glienclmide (Figure 2). The outcome suggests tht dietic control group severely cusing hyperglycemi s compred to the norml control group. Compring with the different doses of UFG treted group rts significntly lowered the elevted lood glucose level (Figure 3). During this investigtion elevtion of fsting plsm glucose level in dietic control group rts t the end of the 28 dy experimentl period significntly (P <.1) oserved. Different doses of UFG nd glienclmide treted dietic rts showed significnt (P <.1) reduction of initil fsting plsm glucose level nd incresing the serum insulin level (Figure 4). Thus, the possile mechnism of ction of UFG is potentiting the insulin y incresing either the pncretic secretion of insulin from the existing cells or y relesing the ound form. Type I nd Type II dietes ptient suffered from therosclerosis (Coronry rtery disese) which my cuse the deth [32]. Not only the therosclerosis nd other fctor like hypertriglyceridemi, hypercholesterolemi nd hypertension my GPx (nmole/mg of protein) Figure 15 Effect of umelliferone β-d-glctopyrnoside on level of GPx (Glutthione peroxidse) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

15 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 14 of SOD (U/mg of protein) Figure 16 Effect of umelliferone β-d-glctopyrnoside on level of SOD (Superoxide dismutse) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). contriute the coronry rtery disese [33-35]. The lipid normlity is circulting dietes nd glucose intolernce (hving tendency to develop the dietes) ptient y insensitivity of peripherl tissue of insulin [36]. In STZ induced dietic rt significnt (P <.1) increse level of totl cholesterol, totl triglyceride, totl VLDL cholesterol nd decrese levels of HDL cholesterol ws oserved. The outcome of the experiment revels tht continue doses of UFG dministered to nimls for 28 dys nerly normlized the lipid profile in dietic induced group rts. Different doses of UFG treted dietic rts, shows mrked reduction in totl cholesterol (Figure 5), totl triglyceride (Figure 6), totl HDL cholesterol ((Figure 7), LDL cholesterol (Figure 8) nd elevte the level of VLDL cholesterol (Figure 9) showed the hypolipidemic effect of UFG. The most effective doses of UFG 4 mg/kg not only decreses the level of TC, TG, nd LDL ut lso improved the crdioprotective lipid HDL. In norml metolism insulin ctivtes the enzyme lipoprotein lipse enzyme nd triglyceride hydrolysis. In dietic condition deficiency of insulin inctivted the oth enzyme nd cuses the hypertriglyceridemi [37]. Another mechnism of ction of UFG my e incresing the level of liver enzyme. The liver plys n importnt role in postprndil hyperglycemi nd the synthesis of glycogen. Hexokinse, glucose-6-phosphte nd fructose- 1-6-iphosphtse re the enzyme found in the liver nd they convert glucose into energy, utilize the glucose, synthesis of glycogen etc. In the liver, hexokinse convert glucose into glucose-6-phosphtse [38,39]. In STZ induced dietic rts inhiit the synthesis of glycogen nd inhiit the level of hexokinse. Decresing the level of hexokinse inhiits the conversion of glucose into glucose-6- phosphte nd incresing the level of glucose in the lood [4]. The STZ induced dietic rts treted with different doses of UFG nd glienclmide hs incresed the level of hexokinse nd increse the utiliztion of glucose to energy conversion (Figure 1). In n erlier discussion it shows tht glucose-6-phosphte regultes the glucose metolism with the help of hexokinse. In STZ induced rts incresed the level of glucose-6-phosphte, incresing levels of glucose-6-phosphte improves the ctivity of gluconeogenetic enzyme nd enhnce the mnufcturing of fts from crohydrtes, due to excess mnufcturing of fts, it strts deposition on kidney nd liver [41,42]. STZ induced dietic rts treted with different doses of UFG nd glienclmide hd normlized the ctivity of glucose-6-phosphtse enzyme ner to norml control y 15 CAT (U/mg of protein) 1 5 c Figure 17 Effect of umelliferone β-d-glctopyrnoside on level of CAT (Ctlse) cholesterol t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns).

16 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 15 of Body Weight (gm) Figure 18 Effect of umelliferone β-d-glctopyrnoside on ody weight t different concentrtions on STZ induced dietic rts, compred to dietic control group rts; vlues re men ± SEM; n = 6; c P <.5; P <.1; P <.1; P >.5 is considered s non-significnt (ns). decresing the enhnced level of glucose-6-phosphte. Another vitl liver enzyme fructose-1-6-iphosphte ply n importnt role in the glycolysis (conversion of glucose into energy) [43,44]. STZ induced dietic group rt showed the increse level of fructose-1-6-iphosphte. Due to increse level of the fructose-1-6-iphosphte decline the glycolysis nd stop the conversion of glucose into energy. STZ induced dietic groups treted with different doses of UFG nd glienclmide decresing the elevted level of fructose-1-6-iphosphte nd rought ck to norml level (Figure 12). STZ induced dietic rts showed increse the level of glucose in the lood which dds to the RBC (red lood cells) in the N terminl of hemogloin chin nd strts the production of glycted hemogloin. The level of glycted hemogloin ws incresed up to 16% in dietes mellitus [45]. Sometime glycted hemogloin cn e used s n indictor of metolic control of dietes Figure 19 Umelliferone β-d-glctopyrnoside (UFG) effect on liver in different groups of rts: (A) Norml control: Norml control group did not produce ny chnges in histopthology (B) Dietic control: Dietic control group rt shown enlrged of micro droplet of ft (yellow rrow) (C) UFG I (1 mg/kg): Tretment with UFG dose (1 mg/kg) shown some prt hving micro droplet of ft deposition (yellow rrow). (D) UFG II (2 mg/kg): Tretment with different dose UFG (2 mg/kg) shown few prticle of micro droplet of ft in liver histopthology (yellow rrow). (E) UFG III (4 mg/kg): Histopthology of UFG (4 mg/kg) drug not showing ny ft deposition nd other ssortments. (F) Glienclmide (1 mg/kg): Stndrd drug treted group shown histopthology similr to the norml control groups. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 1. Figure 2 Effect of umelliferone β-d-glctopyrnoside (UFG) on liver in different groups of rt: (A) Norml control: Norml control group Shown norml histopthology (B) Dietic control: In histopthology of dietic control group rt shown overlown of micro droplet of ft (yellow rrow indicte) (C) UFG I (1 mg/kg): Histopthology of UFG dose (1 mg/kg) shown deposition of some micro droplet of ft (yellow rrow). (D) UFG II (2 mg/kg): In the histopthology of dose UFG (2 mg/ kg) showed few micro droplet deposition of ft (yellow rrow). (E) UFG III (4 mg/kg): Histopthology of UFG (4 mg/kg) drug not showing ny ft deposition nd other chnges. (F) Glienclmide (1 mg/kg): Histopthology of glienclmide treted drug group shown similr to the norml control groups. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 4.

17 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 16 of 19 Figure 21 Effect of umelliferone β-d-glctopyrnoside (UFG) on histopthology study of kidney in different groups of rt: (A) Norml control: Norml control group rt histopthology shown norml size of glomerulus (B) Dietic control: Dietic control rt histopthology shown inflmmtory cell in lood vessels (lue rrow) nd deposition of fts (yellow rrow) (C) UFG I (1 mg/kg): Tested drug UFG (1 mg/kg) treted group rt histopthology shown inflmmtion in lood vessels (rown rrow) nd ft deposition (yellow rrow) (D) UFG II (2 mg/kg): Dose UFG (2 mg/kg) treted group rt histopthology shown only ft deposition (yellow rrow) no inflmmtory lood cells. (E) UFG III (4 mg/kg): Tretment with dose UFG (4 mg/kg) group rt histopthology shown kidney histopthology like the glienclmide treted group (F) Glienclmide (1 mg/kg): Histopthology of Glienclmide treted group rt shown the norml kidney. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 1. memrne ound enzymes nd receptors [48]. The role of nturl nd synthetic ntioxidnt is ltertion of this dmge. The MDA (n indictor of LPO) incresed the level in dietic rt (Figure 14). STZ induced dietic rt groups treted with different doses of UFG significntly decresed the level of MDA. Increse level of GPx (Glutthione Peroxidse) which led to dectivtion of LPO rections (Figure 15). Another primry enzyme such s SOD is cple of chnging the superoxide rdicl to hydrogen peroxide nd CAT (ctlse) is le to inhiit hydrogen peroxide nd involved in detoxifiction of hydrogen peroxide concentrtions. In our investigtion the SOD, CAT nd GPx level were significntly decresed nd level of MDA ws incresed in the different doses of UFG treted groups (Figures 16 nd 17) [5]. The reduction in the ody weight of dietic rts ws oserved in the throughout study of dietes. The weight ws reduced due to gluconeogenesis (ctolism of proteins nd fts). Dietic condition increses the muscle destruction or degrdtion of structurl proteins in ctolism of fts nd protein [51]. Different doses nd glienclmide treted STZ induced dietes groups rt increses the ody weight nd lso demonstrtes the protective effect ginst the controlling the muscle wsting (Figure 18). since glycohemogloin levels pproch norml vlue in dietes in metolic control. In norml condition glycted hemogloin mkes up % of totl hemogloin nd smll volume of lood glucose. Only 4.5-6% of glycted hemogloin covlently onded to the RBC in hemogloin [46]. In our reserch exertion the level of glycted hemogloin ws elevted more thn 4 times higher in the norml control rts. STZ dietic rts treted with different doses of UFG significntly lowering the higher level of glycted hemogloin (Figure 13), which indicte the improved level of glycemic control. Severl method re involved in rective oxygen species in dietes, such s production of lipid peroxidtion (LPO) nd glucose utooxidtion, protein glyction, formtion of dvnced glyction products nd polyol pthwy [47]. STZ induced dietes destroy the pncretic insulin secreting β-cells nd cuse enhncing the level of rective oxygen species (ROS), increse level of ROS dmging the tissue in the ody. In the production of ROS oxygen free rdicl (polyunsturted ftty cids) ply s significnt role [48,49], ROS rect with ll iologicl sustnces nd cell memrne constituent, led to incresing the level of lipid peroxidtion. Incresed level of LPO impirs memrne function y inhiiting the memrne fluidity nd ltering the ctivity of Figure 22 Effect of umelliferone β-d-glctopyrnoside (UFG) on histopthology of kidney norml nd STZ treted groups rt: (A) Norml control: Averge size of glomerulus shown in the norml group rt histopthology. (B) Dietic control: Inflmmtory cells in lood vessels (lue rrow) nd ft deposition (yellow rrow) shown in the histopthology of dietic control group rt. (C) UFG I (1 mg/kg): Some inflmmtory cell in lood vessels (lue rrow) nd deposition of ft (yellow rrow) found in the histopthology of UFG I (1 mg/kg) treted groups rt. (D) UFG II (2 mg/kg): Only ft deposition (yellow rrow) shown in the histopthology of UFG II (2 mg/kg) treted group rt. (E) UFG III (4 mg/kg): Histopthology of dose UFG (4 mg/kg) treted group rt shown verge size of glomerulrs ut slightly igger in size s compred to the norml control. (F) Glienclmide (1 mg/kg): Histopthology of glienclmide treted group niml shown the histopthology similr to the norml kidney. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 4.

18 Kumr et l. BMC Complementry nd Alterntive Medicine 213, 13:273 Pge 17 of 19 Figure 23 Effect of umelliferone β-d-glctopyrnoside (UFG) photomicrogrphs of histologicl chnges in rt pncres: (A) Norml control: Histologicl structure of norml control group rt pncres showing the norml islet (white rrow) (B) Dietic control: Focl necrosis (yellow rrow) showed in the histopthology of dietic control group rt. (C) UFG I (1 mg/kg): Tretment Histopthology of tested drug rt showing igger size of islet nd focl necrosis (yellow rrow) (D) UFG II (2 mg/kg): Histopthology of tested drug rt showing focl necrosis (yellow rrow) (E) UFG III (4 mg/kg): Histopthology of tested drug rt showing norml size of islet (white rrow) (F) Glienclmide (1 mg/kg): glienclmide treted rt pncres showing norml islet (white rrow). For ech group 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 1. Figure 25 Effect of Umelliferone β-d-glctopyrnoside (UFG) photomicrogrphs of histologicl chnge on hert in different groups of rts: (A) Norml control: Norml control group rt showing norml histopthology. (B) Dietic control: Incresed interstitil spce nd distort the interclted disc (yellow rrow) in dietic control group rt histopthology. (C) UFG I (1 mg/kg): Dose UFG (1 mg/kg) treted group rt showing less interstitil spce nd interclted disc (yellow rrow) (D) UFG II (2 mg/kg): Dose UFG (2 mg/kg) treted group rt showing only spce in interclted disc (yellow rrow). (E) UFG III (4 mg/kg): Dose UFG (4 mg/kg) treted group rt did not showing ny chnges in histopthology of hert. (F) Glienclmide (1 mg/kg): Glienclmide (5 mg/kg) treted drug shown the norml histopthology of hert. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 1. Figure 24 Effect of Umelliferone β-d-glctopyrnoside (UFG) photomicrogrphs of histologicl chnges in rt pncres: (A) Norml control: norml histologicl structure of rt pncres showing norml islet (white rrow) (B) Dietic control: Histopthology of dietic control rt showing focl necrosis (yellow rrow) (C) UFG I (1 mg/kg): Histopthology of tested drug rt showing igger size of islet nd focl necrosis (yellow rrow) (D) UFG II (2 mg/kg): Histopthology of tested drug rt showing focl necrosis (yellow rrow) (E) UFG III (4 mg/kg): Histopthology of tested drug rt showing norml size of islet (white rrow) (F) Glienclmide (1 mg/kg): glienclmide treted rt pncres showing norml islet (white rrow). For ech group 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 4. Figure 26 Effect of Umelliferone β-d-glctopyrnoside (UFG) photomicrogrphs of histologicl on hert in different groups of rts: (A) Norml control: Histopthology of norml control group rt norml histopthology of hert (B) Dietic control: Histopthology of dietic control group rt shown incresed interstitil spce nd distort the interclted disc (yellow rrow) (C) UFG I (1 mg/kg): Histopthology of tested drug shown decresed interstitil spce nd interclted disc (yellow rrow) (D) UFG II (2 mg/kg): Histopthology of tested drug shown less interstitil spce (yellow rrow) (E) UFG III (4 mg/kg): Histopthology of tested drug shown norml hert like the glienclmide (F) Glienclmide (1 mg/kg): Histopthology of glienclmide treted drug shown the norml histopthology of hert. The smples were otined from the sme liver ntomicl regions. For ech group, 6 rts were exmined nd 8 pictures were tken. The ove picture for ech group ws chosen rndomly from the 8 pictures in this group. Originl mgnifiction, 4.