R. SERAIRI BEJI 1,2, I. BETTAIEB REBEY 2 *, S. JAMELEDDINE 3, R. KSOURI 2

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1 Assessment of the ntidibetic, ntihyperlipidemic nd ntioxidnt properties of Trigonell foenum-grecum Linneus, 1753 (Fenugreek) in lloxn-induced dibetic rts R. SERAIRI BEJI 1,2, I. BETTAIEB REBEY 2 *, S. JAMELEDDINE 3, R. KSOURI 2 1 Ecole Supérieure des Sciences et Techniques de l Snté de Tunis, BP 176, Bb Souik, Tunisie. 2 Lbortoire des Plntes Aromtiques et Médicinles, Centre de Biotechnologie de Borj-Cédri, BP 901, 2050 Hmmm-Lif; Tunisie. 3 Déprtement de Physiologie, Fculté de Médecine de Tunis; Tunisie. *Corresponding uthor: rosiness@yhoo.fr Abstrct - Fenugreek (Trigonell foenum-grecum Linneus, 1753) is used in lterntive medicine for treting dibetes mellitus nd its ssocited symptoms. The im of this study ws to investigte the ntidibetic, ntihyperlipidemic nd ntioxidnt ctivities of fenugreek in lloxn induced dibetic rts. Dibetes ws confirmed fter 3 dys of single subcutneous injection of lloxn (120 mg/kg) in lbino Wistr rts. Animl s stndrd diet ws supplemented with fenugreek (5%) for 30 dys. Rts were fsted overnight, weighed nd then scrificed on the 31 st dy of the experiment. Their bloods were collected nd submitted to vrious biochemicl mesurements, including blood glucose, totl cholesterol (TC), triglycerides (TG) nd high-density lipoprotein-cholesterol (HDL-CH). Further, low-density lipoprotein-cholesterol (LDL-CH) ws clculted from the dt obtined. Oxidtive enzymes such s superoxide dismutse (SOD), ctlse (CAT) nd glutthione peroxidse (GPx) were ssessed in serum nd pncres homogente. The results showed tht fenugreek supplementtion in dibetic rts significntly decresed the levels of glucose (342.2 ± 23.9 mg/dl vs ± 18.7 mg/dl; p< 0.05), TG (0.87±0.17g/l vs 0.49±0.09; p< 0.05), TC (0.95±0.11g/l vs 0.75±0.17; p<0.05) nd LDL- CH (0.79±0.5g/l vs 0.53±0.15g/l; p<0.05) compred to dibetic control group. Furthermore, fenugreek incresed the level of HDL-CH from 0.11±0.01g/l to 0.14±0.04g/l (p<0.05). These results re ccompnied with improved weight gin of dibetic rts compred with the dibetic controls. Concerning oxidtive stress, fenugreek significntly improved SOD, CAT nd GPx ctivities both in serum nd in pncres homogente. Fenugreek demonstrted significnt ntidibetic, ntihyperlipidemic, nd ntioxidnt ctivities tht my be due to its multiple effects involving both pncretic nd extr-pncretic mechnisms. Fenugreek hs the potentil to be used s dietry supplement for the mngement of dibetes. Keywords: ntihyperglycemic, ntihyperlipidemic, lloxn, ntioxidnt enzymes, fenugreek 1. Introduction Dibetes mellitus is common disese nd its prevlence is incresing worldwide. The World Helth Orgniztion estimtes prevlence of 347 million people with dibetes worldwide in 2013 (WHO, 2013). This prevlence is expected to double between (Wild et l. 2004). Dibetes is chrcterized by hyperglycemi nd disturbnces in crbohydrte, protein, nd lipid metbolism. Chronic hyperglycemi tht occurs in dibetes is ssocited with long-term dmge, dysfunction, nd filure of different orgns, especilly the eyes, kidneys, nerves, hert, nd blood vessels (Americn Dibetes Assocition, 2013). Hyperglycemi my perturb cellulr ntioxidnt defense systems nd dmge cells. Free rdicls re formed disproportiontely in dibetes by glucose oxidtion, nonenzymtic glyction of proteins, nd the subsequent oxidtive degrdtion of glycted proteins. Abnormlly high levels of free rdicls nd the simultneous decline of ntioxidnt defense mechnisms cn led to dmge of cellulr orgnelles nd enzymes, incresed lipid peroxidtion, nd development of insulin resistnce (Henriksen et l. 2011). Dibetes is lso ssocited with profound ltertions in the plsm lipid nd lipoprotein profile nd n incresed risk of premture therosclerosis nd crdic filure (Betteridge et l. 1997). Clerly, hyperlipidemi (high in TC, TG, LDL-CH) nd in vivo oxidtion stress in dibetic ptients deteriorte dibetic interrelted Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

2 complictions. However, compounds tht show ntioxidnt nd nti-inflmmtory potentil my prevent pncretic islet insulin-producing β cells from selective destruction (Jidne et l. 2009), inhibit islet poptosis, dely the occurrence of type 1 Dibetes (Hr et l. 2006) nd llevite dibetic interrelted complictions. Due to numerous injuries nd occsionlly ftl disese cused in dibetic ptients, there is need to find the tretment methods nd wys to reduce nd prevent it. Presently, with regrd to the problems of prepring nd injecting insulin nd other glucose-lowering drugs nd lso considering the side effects of synthetic drugs, the use of herbl medicines hs ttrcted the ttention of reserchers. Fenugreek (Trigonell foenum-grecum Linneus, 1753) is used s condiment, s supplement to bsiss, locl diet preprtion lrgely consumed in Tunisi nd s constituent of the dily diet in severl Africn countries. Since non processed herbs re usully more tolerted thn extrcted or formulted products nd ptients prefer to consume plnts s sld, spice, nd so forth, in this work, we imed to investigte the effect of fenugreek powder supplemented to rt diet on glycemic, lipidemic nd ntioxidnt sttus of dibetic rts. 2. Mterils nd methods 2.1. Plnt mteril Fenugreek seed used in the present study ws purchsed from the locl herbl mrket nd identified by Professor Abderrzek Smoui t Lbortory of Medicinl nd Aromtic Plnts. Fenugreek seeds were clened, dried, finely powdered in grinding mchine. In the present study, powdered fenugreek seeds were mixed with stndrd diet (5%) Animls Thirty-two mle Wistr lbino rts weighing g nd verging 12 weeks old were obtined from Psteur Institute of Tunisi nd used in this study. They were housed in polypropylene rt cges in n niml room with controlled lighting (12 h light/12 h drkness) nd temperture (22 ± 2 C). The nimls were given stndrd pellets diet nd wter d libitum. All the niml experiments were performed ccording to the recommendtions of ethic committee of Tunis University for cre nd Interntionl Council of Lbortory Animl Science. Induction of dibetes ws crried out in rts by dministering subcutneous injection of 120 mg/kg of freshly prepred lloxn solution (lloxn monohydrte, Sigm Chemicls Co.), dissolved in cette buffer (ph 5.5) prepred immeditely before use. The control groups were injected only with the sme volume of cette buffer s the dibetic groups received. Alloxn induces dibetes through destructing Lngerhns islnds of the pncres. Therefore, lrge mount of insulin is relesed from the pncres cells fter the injection. In order to prevent hypoglycemic shock, during the first 24 h fter the lloxn injection, the rts received 10% dextrose insted of wter (Shhidi et l. 2001; Kwon et l. 2003). Induction of dibetes ws confirmed by mesuring fsting blood glucose (FBG) three dys fter lloxn injection. Rts with blood glucose level of 250mg/kg or higher were considered to be dibetic (Ghorbni et l. 2013) Groups of study The experiment ws crried out on four groups; ech group contined 8 rts s follows: Group I. Norml control Group II. Dibetic control Group III. Norml control fed with diet enriched with fenugreek (5%, w/w) Group IV. Dibetic rts treted fed with diet enriched with fenugreek (5%, w/w) The tretment ws initited two dys fter lloxn injection nd continued for 4 weeks. At the end of the study, nimls were fsted overnight nd nesthetized with chloroform. Blood smples were collected from the niml's hert nd the serum ws seprted by centrifugtion (3000 rpm t 40 C for 15 min) nd stored t -30 C for different biochemicl nlyses Estimtion of body weight The body weights in experimentl nimls were determined before the study nd t 7, 14, 21 nd 28 dys fter the study by digitl blnce (Entris, precision 10mg). These weights were determined t the sme time during the morning. Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

3 2.5. Estimtion of blood glucose Throughout the 4-week tretment period, fsting (12 hours) blood glucose ws mesured before the study nd t 7, 14, 21 nd 28 dys fter the study on lterl til vein. Blood smples were nlyzed using n ACCU-Check glucose meter (Roche, Mnnheim, Germny) Estimtion of blood lipid profile Lipid in serum concentrtion including TG, TC nd HDL-CH were determined with the use of commercilly vilble enzymtic kits from Biomghreb Anlyticls (Tunisi). LDL-CH ws estimted by Friedwld method s follows: LDL cholesterol = totl cholesterol- HDL cholesterol- (Triglyceride 5) All ssessment ssys nd kits were performed in ccordnce with the mnufcturer instructions nd protocols Level of ntioxidnt enzymes ctivities in pncres tissue nd serum Pncres tissue homogentes of the control nd experimentl groups of nimls were prepred with 0.1 M Tris HCl buffer (ph 7.4) t 4 C using tissue homogeneiser. The resulting tissue homogentes were used for biochemicl mesurements. The ctivities of three ntioxidnt enzymes: SOD, CAT nd GPx were determined in pncres homogente nd in serum. SOD ctivity ws mesured ccording to the method of Beyer nd Fridovich (Beyer et l. 1987). CAT ctivity ws determined using the method of Aebi (Aebi et l. 1984) nd GPx ctivity ws mesured ccording to the method of Flohe nd Gunzler (Flohe nd Gunzler, 1984) Protein quntifiction in pncres homogente Protein content in pncres smples ws ssyed s described by Lowery et l. (Lowry et l. 1951), using bovine serum lbumin s stndrd Sttisticl nlysis Sttisticl nlysis ws performed with Sttistic TM softwre, using one-wy nlysis of vrince (ANOVA). Sttisticl ssessments of differences between men vlues were performed by Duncn s multiple rnge test t P Results nd discussion 3.1. Acute toxicity in rts Rts dministrted fenugreek did not develop ny clinicl signs of toxicity either immeditely or during tretment nd no mortlity occurred Effects of Fenugreek on body weight nd plsm glucose There ws significnt increse in blood glucose nd decrese in body weight in lloxn induced group II dibetic nimls s compred to the norml ones indicting the impired glucose metbolism. Fenugreek supplemented diet significntly lowered the blood glucose nd elevted body weight when compred with the untreted dibetic group. Tble 1 shows body weight nd blood glucose levels of different niml groups t the beginning nd t the end of the experiment. There ws no significnt difference in body weight nd blood glucose levels between groups before induction of dibetes. At the end of the experimentl period, the body weight of the untreted (249.5±8.3g) nd fenugreektreted dibetic rts (221.5±10.3g) were lower thn control rts (264.3±10.2g). Considering plsm glucose levels, untreted dibetic nimls hd significntly (P <0.05) elevted plsm glucose levels (342.2 ± 23.9mg/dl) compred with control nimls (88.6 ± 4.8mg/dl). Supplementtion diet with fenugreek to dibetic rts decresed the plsm glucose levels of the treted groups (171.0 ± 18.7mg/dl) compred with the untreted dibetic group (171.0 ± 18.7mg/dl; P <0.05). This beneficil effect on glycemic sttus is expected to hppen s it hs been confirmed with repeted studies (Sjd Arshdi et l, 2015; Gddm et l. 2015). Antihyperglycemic effect of medicinl plnts Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

4 is chieved by different mechnisms including decresing glucose bsorption from intestine, enhncing insulin secretion from bet cells, incresing glucose uptke by tissues, inhibiting glucose production in liver, nd incresing pncretic tissue regenertion nd/or presence of insulin-like gents in plnts (Hui et l, 2009; Shnmugsundrm et l. 1981). The ntihyperglycemic effect of fenugreek hs been hypothesized to be due to the minocid 4-hydroxyisoleucine which cts by the enhncement of insulin sensitivity nd glucose uptke in peripherl tissues (Singh et l. 2010). Other components of Fenugreek seeds re known to hve hypoglycemic effects include rginine, tryptophn, scorbic cid, nicin, nicotinic cid, chromium, copper, mgnesium, mngnese, zinc, gentinine, choline nd quercetin (Thkrn et l. 2004). Tble 1: Body weight nd plsm glucose levels of different niml groups t the beginning nd end of the study Groups Plsm glucose (mg/dl) Body weight (g) Beginning End Beginning End Group I 86.6 ± ± ± ±10.2 Group II ± 35.0 b ± 23.9 b 217.5± ±10.2 b Group III 85.6 ± ± ± ±8.3 Group IV ± 29.8 b ± 18.7 c 210.0± ±10.3 c Dt re expressed s men ± SD of mesurements from 8 rts. Groups re lbelled s follows: Group I: norml control, Group II: dibetic control, Group III: Norml control fed with diet enriched with fenugreek (5%, w/w), Group IV: Dibetic rts treted fed with diet enriched with fenugreek (5%, w/w). Different letters in the sme rw indicte significnt differences t P 0.05 s determined by Duncn s multiple rnge tests Effect of fenugreek on serum lipids Dibetes is often ssocited with dyslipidemi, min risk fctor of crdiovsculr diseses. Therefore, the levels of serum triglyceride nd cholesterol re usully elevted in dibetic ptients (Ptel et l. 2008) Here, we investigted the effect of diet contining 5% fenugreek on lipidemic sttus of dibetic rts. Tble 2 shows the level of serum lipids in study groups. In ddition to reducing blood glucose, n insulin-mimetic gent should be ble to normlize dyslipidemi. There ws significnt elevtion in the level of TG (0,87±0,17 g/l versus 0,45±0,07 g/l, P <0.05) nd TC (0,95±0,11 g/l versus 0,60±0,07g/l, P < 0.05) in dibetic control rts s compred with norml group. Our results showed tht fenugreek powder supplemented to rt diet extrct t dose of 5 % ws ble to cuse significnt decrese in TG nd TC levels in niml serum. The levels of TG nd TC in fenugreek treted group were 0,49±0,09 g/l versus 0.40±0.11g/l in dibetic control group nd 0,75±0,17g/l versus 0.63± 0.09 g/l in dibetic control group, respectively. The hyperlipidemi observed in the untreted dibetic rts in the present study could indicte n increse in the mobiliztion of free ftty cids from the peripherl ft depots. This could result from the uninhibited ctions of lipolytic enzyme lipse cused by insulin deficiency chrcteristic of the dibetic stte (Omolol et l. 2014). The incresed levels of CH nd TG were brought bck to ner norml by the tretment with fenugreek. This observed restortion of the lloxn evoked chnges in the serum lipid profile shows the protective nture of fenugreek. Nevertheless, the lipid-lowering compounds in fenugreek seeds could be of polyphenolic nture. Indeed, Wilox et l. (2001) provided evidence tht nringenin not only decrese cholesterol biosynthesis but lso inhibit cyl trnsferse (ACAT), key enzyme involved in the esterifiction nd bsorption of cholesterol, secretion of heptic LDL-CH, nd cholesterol ccumultion in the rteril wll. Nringenin is in fct well known flvonoid which ws detected in fenugreek extrct (Belid- Nouir et l. 2012). Others compounds were lso detected like 4-hydroxyisoleucine, n typicl brnched-chin mino cid derived from fenugreek (Jetté et l. 2000) lso detected in fenugreek (Belid-Nouir et l. 2012). This effect could explin the significnt increse in plsmtic HDL-CH following TC decrese when fenugreek seeds re dministrted to lloxn-treted rts. Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

5 Numbers of studies hve shown tht steroid sponin extrcted from fenugreek seeds hs the bility to modify cholesterol sttus by its cpcity to bind both cholesterol nd bile cids (Bsu et l. 2010). Diosgenin, steroidl spogenin extrcted from fenugreek seeds, hs in fct been shown to reduce TC s well s LDL-CH in high-cholesterol fed quils (Al-Mtubsi et l. 2011). On the other hnd, trigonelline, n lkloid isolted from fenugreek seeds, ws found ble to normlize the rte of lipogenesis in streptozotocin induced hyperglycemic rts by stimulting heptic lipogenic enzymes (El-Soud et l. 2007). Tble 2: Comprtive levels of serum triglycerides, cholesterol, HDL-CH nd LDL-CH in different rt groups Groups Serum lipids (g/l) Triglycerides Cholesterol HDL-CH LDL-CH Group I 0,45±0,07 0,60±0, ± ±0.11 Group II 0,87±0,17 b 0,95±0,11 b 0.11±0.10 b 0.79±0.50 Group III 0,40±0,11 0,63±0, ± ±0.13 Group IV 0,49±0,09 0,75±0,17 c 0.14± ±0.15 Dt re expressed s men ± SD of mesurements from 8 rts. Groups re lbelled s follows: Group I: norml control, Group II: dibetic control, Group III: Norml control fed with diet enriched with fenugreek (5%, w/w), Group IV: Dibetic rts treted fed with diet enriched with fenugreek (5%, w/w). Different letters in the sme rw indicte significnt differences t P 0.05 s determined by Duncn s multiple rnge tests Effect of Fenugreek on pncretic nd plsmtic oxidtive enzymes The common chronic dmge in dibetes ptients is closely relted to elevted oxidtive/inflmmtory ctivities with continuum of tissue insults leding to more severe crdiometbolic nd interrelted complictions (Ndisng et l. 2010). In the present study, we lso investigted the ntioxidnt effects of fenugreek in plsm nd homogente pncres of lloxn-induced dibetic rts. To our knowledge, no studies compred oxidtive enzymes ctivities in plsm nd tissues. Our results show tht untreted dibetic rts showed significntly reduced SOD (p<0.05), GPx (p < 0.05) nd CAT (p < 0.05) ctivities in plsm nd pncres homogente, compred to the non-dibetic control group. The enriched diet with fenugreek for four weeks produced significnt increses in SOD (p < 0.05), CAT (p < 0.05) nd GPx (p < 0.05) ctivities both in plsm nd in pncres homogente. The oxidtive stress induced by lloxn rises due to compromise in nturl ntioxidnt mechnism nd n increse in oxygen free rdicl production (El-Demerdsh et l. 2005). Indeed, free rdicl scvenging enzymes such s SOD nd CAT protect the biologicl system from oxidtive stress (Del Rio et l. 2005). SOD ctlyses the rection in which superoxide nion is converted to hydrogen peroxide nd oxygen while CAT is hem-contining ubiquities enzyme tht detoxifies H 2 O 2 into wter nd oxygen (Selvn et l. 2008). The reductions observed in the ctivities of SOD nd CAT in the dibetic control group suggest their excessive utiliztion in ttenuting free rdicls generted during the metbolism of lloxn. This observtion hs lredy been reported in dibetic nimls (Onyek et l. 2013). Increse in their ctivities is n indiction of their bility to scvenge ROS, thus contributing to the protective effect ginst oxidtive stress nd preventing further dmge to membrne lipids. Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

6 Tble 3: Effect of fenugreek nd/or lloxn tretment on SOD, CAT nd GPx ctivities in plsm nd pncretic homogentes of treted rts Pncretic homogente Plsm Groups SOD U/mg protein CAT (10-3 mm.min - 1.mg -1 protein) GPx (Mol H 2 O 2.min - 1.mg -1 protein) SOD U/mg protein CAT (10-3 mm.min - 1.mg -1 protein) GPx (Mol H 2 O 2.min - 1.mg -1 protein) Group I Group II Group III Group IV 20.2± ±1.2 b 23.6± ± ± ± ± ±2.9 b 0.6±0.2 b ±5.07 b 51.5± ± ± ±4.9 c 1.2±0.2 c ± ± ± ±0.26 b ±30.27 b 4.46±0.44 c ±58.3 c 3.82± ±312.5 c Dt re expressed s men± SD of mesurements from 8 rts. Groups re lbelled s follows: Group I: norml control, Group II: dibetic control, Group III: Norml control fed with diet enriched with fenugreek (5%, w/w), Group IV: Dibetic rts treted fed with diet enriched with fenugreek (5%, w/w). Different letters in the sme rw indicte significnt differences t P 0.05 s determined by Duncn s multiple rnge tests. 4. Conclusion In conclusion, the present study demonstrted tht the hypoglycemic nd ntihyperlipidemic ctivities exhibited by fenugreek were effective enough to llevite lloxn-induced dibetes in experimentl rts. The beneficil effect of diet supplemented with fenugreek is presumbly ttributed to its potent hypoglycemic nd ntihyperlipidemic properties, s well s ntioxidnt potentil. Further studies using different doses nd for different periods re obviously needed to determine the dose required on the protective effects of fenugreek in humns nd to mke its use suitble s n effective functionl food with therpeutic potentil. 5. References Aebi H (1984) Ctlse in vitro. Methods Enzymol 105: Al-Mtubsi HY, Nsrt NA, Oriqut GA, Abu-Smk M, Al-Mzin KA, Slim M (2011) The hypocholesterolemic nd ntioxidtive effect of dietry diosgenin nd chromium chloride supplementtion on high-cholesterol fed Jpnese quils. Pk J Biol Sci 14: Americn Dibetes Assocition (2013) Dignosis nd clssifiction of dibetes mellitus. Dibetes Cre. 36: Bsu TK, Srichmroen A (2010) In: Bioctive Foods in Promoting Helth. Wtson RR, Preedy VR, editor. Sn Diego: Acdemic Press; Helth Benefits of Fenugreek (Trigonell foenum-grecum leguminosse) pp Belïd-Nouir Y, Bkht1 H, Bouziz M, Flehi-Slim I, Hous H, Ben Cheikh H (2012) Study of lipid profile nd prieto-temporl lipid peroxidtion in AlCl3 medited neurotoxicity modultory effect of fenugreek seeds. Lipids Helth Dis 11:16 Betteridge J (1997) Lipid disorders in dibetes mellitus. In: Pickup JC, Willims G, editors. Text Book of Dibetes. London: Blckwell Science Publishers. Beyer WF, Fridovich I (1987) Assying for superoxide dismutse ctivity: some lrge consequences of minor chnges in conditions. Anl Biochem 161: Del Rio D, Stewrt AJ, Pellegrini N (2005) A review of recent studies on mlondildehyde s toxic molecule nd biologicl mrker of oxidtive stress. Nutr Metb Crdiovsc Dis 15: Deshpnde AD, Hrris-Hyes M, Schootmn M (2008) Epidemiology of dibetes nd dibetes-relted complictions, Phys Ther 88: El-Demerdsh FM, Yousef MI, El-Ng NI (2005) Biochemicl study on the hypoglycemic effects of onion nd grlic in lloxn-induced dibetic rts. Food Chem Toxicol 43: Seriri Beji et l. (2016) / Journl of new sciences, Agriculture nd Biotechnology, 28(7),

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