Effects of yam dioscorin interventions on improvements of the metabolic syndrome in high-fat diet-induced obese rats

Transcription

1 Shih et l. Botnicl Studies (215) 56:4 DOI /s RESEARCH Effects of ym dioscorin interventions on improvements of the metolic syndrome in high-ft diet-induced oese rts Shen-Ling Shih 1, Yin-Shiou Lin 2, Shyr-Yi Lin 3,4* nd Wen-Chi Hou 2,5,6* Open Access Astrct Bckground: The metolic syndrome (MS) is termed cluster of multiple metolic risk criteri which is positively correlted with crdiovsculr disese nd type 2 dietes mellitus (DM). Ym dioscorins hve een reported to exhiit iologicl ctivities, however, little is known their preventive effects on the MS. Therefore, high-ft (HF) diet ws used to induce Wistr rt oesity nd then ym dioscorin (5 mg/kg, dio5) ws intervened dily concurrent ( + dio5) for five weeks to check the chnges of weights of ody nd tissues, lood pressures, nd impired glucose tolernces. The in vitro peptic hydrolystes of dioscorin with moleculr mss etween 3 kd nd 1 kd nd less thn 3 kd were used to determine dipeptidyl peptidse IV (DPP IV) inhiitory ctivities which DPP IV inhiitor hs een reported to prevent nd tret type 2 DM. Results: There were no significnt difference in ody weights, feed intkes, feed conversion, nd weights of dipose tissues of oese rts in groups of HF nd ( + dio5). However, the systolic lood pressures in oese rts of 2-, 3- nd 4-week dioscorin interventions were showed significntly lower (P <.5) compred to the HF group. The dioscorin intervention (HF+ dio5) ws showed significntly different (P <.5) nd improved the impired glucose tolernces compred to HF group in oese rts y the orl glucose tolernce tests. It ws lso found tht the frction with different moleculr mss of dioscorin peptic hydrolystes (5 mg/ml) showed inhiitory ctivities ginst DPP IV using sitgliptin phosphte s positive controls. Conclusions: Ym dioscorins exhiit improved MS ctivities in oese rts which the relted mechnisms my need further investigtions. Keywords: Dioscorin; Dipeptidyl peptidse IV (DPP IV); Metolic syndrome (MS); Peptic hydrolystes; Systolic lood pressure; Ym Bckground The metolic syndrome (MS) is termed cluster of five multiple metolic risk criteri, including (1) dominl oesity (centrl oesity), (2) hypertriglyceridemi [fsting triglycerides (TGs) 15 mg/dl], (3) low serum high-density lipoprotein cholesterol (HDL 4 mg/dl), (4) elevted lood pressure (systolic 13 mmhg or distolic 85 mmhg), nd (5) high fsting * Correspondence: sylin@tmu.edu.tw; wchou@tmu.edu.tw Equl contriutors 3 Deprtment of Primry Cre Medicine, Tipei Medicl University Hospitl, Tipei, Tiwn 2 Grdute Institute of Phrmcognosy, Tipei Medicl University, Tipei, Tiwn Full list of uthor informtion is ville t the end of the rticle lood glucose ( 11 mg/dl), nd n incresing oesity nd sedentry lifestyles relte positively the rising MS worldwide prevlence (Dy 27; Alerti et l. 29). A generl dignosis with three out of ove-mentioned five criteri cn recognize s the MS (Dy 27). Insulin resistnce nd impired glucose tolernce re lso two importnt metolic risk criteri which my involve in fctors for the MS generl dignosis (Dy 27). The helth risks of MS ssocited with oesity vry mong individuls, ut consistently include type 2 dietes mellitus (type 2 DM), hypertension, coronry hert disese nd cncer (Zimmet 1982; Prentice 26). The dominl oesity my ply the centrl role in MS which is the sitution of exceeding viscerl ft deposited in peritonel cvity, nd will initite inflmmtion nd dyslipidemi, increse the lood pressure 215 Shih et l.; licensee Springer. This is n Open Access 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 credited.

2 Shih et l. Botnicl Studies (215) 56:4 Pge 2 of 9 nd decrese insulin sensitivity ccompnied with norml lood glucose. Oesity is lmed s mjor contriuting fctor in over.3 million deths per yer in the Americ nd relted economic costs over US 1 illion per yer (Dniels 26; Rodgers et l. 212). In fct, oesity ssocited with dietes re considered not only clinicl prolem ut lso pulic helth issue in mny countries, nd 8% of overweight people re lso dignosed with type 2 DM which re referred s the twin epidemics (Smyth nd Heron 25). Severl niml models re estlished nd suitle for one or more metolic risk criteri in MS studies (Pnchl nd Brown 211), such s genetic rodent models, including d/d mice, o/o mice, Zucker dietic ftty rts, nd Otsuk Long-Evns Tokushim ftty (OLETF) rts etc., however, such genetic muttions in estlished rodent nimls re few reported in humns; on the other hnd, the diet-induced rodent models my mimic closely to MS symptoms in humns, including fructose-induced, sucroseinduced, nd high-ft (HF) diet induced rodent nimls (Pnchl nd Brown 211). The s re reported to induce overweight, oesity, dyslipidemi, insulin resistnce, nd high lood pressures in rodent nimls (Aguli nd Mndrim-de-Lcerd 23; Woods et l. 23; Koysi et l. 21), nd re widely used for MS study (Frigolet et l. 213). The dipeptidyl peptidse IV (DPP IV) is serine-type proteinse (EC ) which metolizes peptide hormones, such s glucgon-like peptide-1 (GLP-1), n insulinotropic peptide hormone cn stimulte glucosedependent insulin secretions (Mentlein 25; Drucker 26; Idris nd Donnelly 27). DPP IV inhiitors tht control the glycermi y modulting the GLP-1 levels re currently developed for type 2 DM tretments (Smyth nd Heron 25; Idris nd Donnelly 27). DPP IV cleves the Pro or Al t the second position of the ctive N-terminl GLP-1(7 37) or GLP-1(7 36) mide which results in functionl inctive GLP-1(9 37) or GLP-1(9 36) mide (Drucker 26; Idris nd Donnelly 27). Therefore, reserchers ttempted to isolte potentil DPP IV inhiitory peptides from protein hydrolystes, such s pepsin-pncretin hydrolystes of sodium cseinte, skim milk powders nd milk protein concentrtes (Lcroix nd Li-Chn 212), Ummizyme G hydrolystes of deftted rice rn (Htnk et l. 212), nd pepsin-treted whey protein (Lcroix nd Li-Chn 213). Yms re recognized s herl plnts since the tuer dried slices re widely used s Chinese herl medicines, nd the fresh tuer hs lso een stple food in West Afric, Southern Asi, nd the Crien. The tuers of ym storge protein, dioscorin, ccount for out 9% of the extrctle wter-solule proteins from different species s estimted y the immune stining method (Hou et l. 2). The ym dioscorin nd/or its peptic hydrolystes hve een reviewed for iologicl ctivities in vitro nd/or in vivo (Lu et l. 212), mong which the ntihypertensive ctivities (Hsu et l. 22; Lin et l. 26; Liu et l. 29,; Lin et l. 214) nd ntioxidnt ctivities (Hou et l. 21; Liu et l. 26; Hn et l. 213, 214,,c) my involve in MS studies. Therefore, which the ft composition provide 6% of totl clories is used to induce oese rts nd then ym dioscorin is intervened to oserve the chnges of ody weights, lood pressures, nd glucose tolernces. It is lso to test the pepsin hydrolystes of ym dioscorin to evlute DPP IV inhiitory ctivities. Methods Mterils DPP IV (lyophilized powder, 1 units/mg protein, D- 752), glucose, Gly-Pro p-nitronilide, pepsin (346 units/ mg solid, P-6887), nd sitgliptin phosphte were purchsed from Sigm Chemicl Co. (St. Louis, MO). The HF diet for oesity induction (ft composition provide 6% of totl clories, D12942) ws purchsed from Reserch Diets, Inc. (NJ, USA). The stndrd mouse/rt chow (ft composition provide % of totl clories, Prol RMH25, 5P14 Diet; PMI Nutrition Interntionl, MO, USA). s-induced oese rts for MS studies Mle 1-week-old Wistr rts (N = 24) were purchsed from Ntionl Lortory Animl Center (Tipei, Tiwn) nd housed in wire-ottomed stinless steel cges in temperture- nd humidity-controlled room (t 22 C) with 12-h light/drk cycle which hd free ccess to the feeds nd wter. All niml experimentl procedures were reviewed nd pproved y the Institutionl Animl Cre nd Use Committee, Tipei Medicl University (LAC-1-38). After cclimtion for one week, rts were rndomly divided into three groups (N = 8 for ech group), including lnk group for stndrd mouse/rt chow nd two HF diet-induced oese groups (one control s HF group nd one dioscorin-intervened group) for 7-dys. In the dioscorin intervened group, ym dioscorin (5 mg/kg, dio5) ws intervened dily concurrent s (HF + dio5) from dy-36 to dy-7. The rt weights nd feed intkes were recorded during experiments. The feed conversion is defined s rtio of mounts of feed intkes (g) divided y rt weight gins (g) during experimentl periods. Rts were scrificed, orgns (hert, lung, liver, kidney, nd spleen) nd dipose tissues (retroperitonel ft, mesenteric ft, viscerl ft, nd testicle ft) were collected nd weighted for comprisons. Chnges of lood pressures in the dioscorin intervention of -induced oese rts The systolic lood pressure (SBP) nd distolic lood pressure (DBP) of rts were mesured t the end of ech

3 Shih et l. Botnicl Studies (215) 56:4 Pge 3 of 9 week fter dioscorin intervention for 1-, 2-, 3-, nd 4- weeks y using n indirect til-cuff lood pressure meter (BP-98A, Softron Co. Ltd. Tokyo, Jpn). Distilled wter (.5 ml) ws dministered to the rts in the norml diet group (the lnk) nd HF group (the control) insted of dioscorin solutions for comprisons. Impired glucose tolernces in the dioscorin interventions of -induced oese rts y orl glucose tolernce tests Impired glucose tolernce of -induced oese rts were mesured y orl glucose tolernce tests (OGTT) following the previous report (Ito et l. 21; Andrikopoulos et l. 28) with modifictions. Rts of ech group t the ends of the 1 th -week were fsted for 16 hours nd glucose ws dministered y orl gvge t 1 g/kg ody weight. Blood (.1 ml) ws otined from the til vein of rts t, 5, 3, 6, 9, nd 12 min fter the glucose lods. Plsm glucose were determined y using RANDOX glucose kit (Rndox Lortories-US, Ltd. USA). The ssy principle is sed on the hydrogen peroxide genertion ctlyzed y glucose oxidse nd further rected with phenol nd 4-minophenzone to produce reddish violet quinoneimine dye with sornce t 55 nm. DPP IV inhiitory ctivities of peptic hydrolystes of ym dioscorin The procedure for peptic hydrolystes of ym dioscorin ws following the previous reports (Hsu et l. 22; Liu et l. 26). The hydrolytic rtio (wt/wt) of ym dioscorin (g) to pepsin (g) ws set t 5/1 in.1 M KCl uffer (ph 2.) with stirring t 4 C for 3 dys. After hydrolysis, 1. M Tris HCl uffer (ph 8.3) ws dded to ph 7.5 to stop hydrolysis. The moleculr cut-off centricon or centriprep device(ym-1,1kd;ym-3,3kd;milliporeco.,usa)ws used to seprte peptic hydrolystes of ym dioscorin into three portions s followings: higher thn 1 kd (peptide > 1 kd), etween 3 to 1 kd (3 kd < peptide < 1 kd), nd less thn 3 kd (peptide < 3kD). The frctions of etween 3 to 1 kd (3 kd < peptide < 1 kd) nd less thn 3 kd (peptide < 3kD) were further nlyzed y reverse-phse C18 Sphericl HPLC column (1 mm 25 mm). The moile phse ws mixed in stepwise grdients with solvent A (deionized wter contining.1% trifluorocetic cid) nd solvent B (1% cetonitrile contining.1% trifluorocetic cid) s followings, to 5 min, 1% to 79% solvent A nd to 21% solvent B; 21 min, 75% solvent A nd 25% solvent B; 22 min to 27 min, 6% solvent A nd 4% solvent B; 4 min, % solvent A nd 1% solvent B. Flow rte ws 3 ml/min. The detector ws set t 22 nm. The ltter two frctions were lyophilized for DPP IV inhiitory ssys. The DPP IV inhiitory ctivity ws ssyed ccording the previous report (Lcroix nd Li-Chn 213) with modifictions. The DPP IV enzyme powder ( 1 units/mg protein) ws re-dissolved in 1 ml of 1 mm Tris uffer (ph 8.) s stock enzyme solution nd 5-fold dilution efore uses s working enzyme solution. The sustrte, Gly-Pro p-nitronilide, ws prepred s 4 mm stock solutions. The sitgliptin phosphte, the DPP IV inhiitor (Drucker et l. 27) s the positive control, ws prepred s 1 μm stock solutions. Ech 5 μl of working DPP IV enzyme solution nd peptic dioscorin frctions ws mixed t 37 C for 1 min, then 5 μl of sustrte ws dded nd djusted to 2 μl y 1 mm Tris uffer (ph 8.). The sitgliptin phosphte ws used insted of tested dioscorin frction for the control. The 1 mm Tris uffer (ph 8.) ws used insted of tested dioscorin frction for the lnk. The sornce t 45 nm ws mesured t 6 min y using n ELISA reder (TECAN Sunrise microplte reder; Männedorf, Switzerlnd). The DPP IV inhiition (%) ws clculted s (A45 lnk -A45 smple or control / A45 lnk ) 1%. Sttisticl nlyses Dt were expressed s men ± SEM in the rt weight chnges nd impired glucose tolernces y OGTT methods, others were expressed s men ± SD. Multiple group comprisons under the sme treted time were performed using one-wy nlysis of vrince (ANOVA) followed y the post hoc Tukey s test, nd vlues tht hve not een indicted with the sme lphet were significntly different (P <.5). Sttisticl nlysis ws performed using the GrphPd Prism 5. softwre (Sn Diego, CA, USA). Results Effects of dioscorin interventions on ody weights of HF diet-induced oese rts At the dy 36, the verge weight of rts in the group of norml diet,, nd ( + dio5) ws ± (g), ± 11.6 (g), nd ± (g), respectively. At the dy 68, the verge weight of rts in the group of norml diet,, nd ( + dio5) ws ± 18.2 (g), ± (g), nd ± (g), respectively. Rts fed with the stndrd chow (the norml diet) showed lighter verge ody weights nd significnt difference (P <.5) compred to those fed with HF diets without or with dioscorin interventions during 7- dy experiments (Figure 1A). While, the verge weight of rts in two groups of HF-induced oese rts, nmely HF diet group or with ym dioscorin interventions ( + dio5) group showed no significnt differences (P >.5) t the eginning (dy 36) nd the ends (dy 68) of experiments (Figure 1A). It ment tht ym dioscorin interventions t 5 mg/kg for 5 weeks showed no weight reduction ctivity ginst oese-induced rts t the present protocol designs. Figure 1B showed the feed

4 Shih et l. Botnicl Studies (215) 56:4 Pge 4 of 9 Weight (g) Feed intke (g) (A) dioscorin intervention +dio5 Norml diet (B) Dy Dy +dio5 Norml diet Feed conversion Weight rtio (% of ody weight) (C) (D) hert lung liver +dio5 Norml diet Dy +dio 5 Norml diet kidney spleen retroperitonel ft mesenteric ft viscerl ft testicle ft Figure 1 Effects of dioscorin interventions (5 mg/kg) on (A) ody weights, (B) feed intkes, (C) feed conversion (feed intkes/weight gins), nd(d) weights of orgn nd ft tissues of high ft-diet induced oese rts. Arrow indictes the dioscorin intervention from dy 36 to dy 7. Dt were expressed s men ± SEM in the rt weight chnges nd others were expressed s men ± SD. Multiple group comprisons under the sme treted time were performed using one-wy nlysis of vrince followed y the post hoc Tukey s test, nd vlues tht hve not een indicted with the sme lphet were significntly different (P <.5). HF, high-ft diet; ( + dio5), ym dioscorin (5 mg/kg, dio5) ws intervened dily concurrent. intkes during experiments. The feed intkes in three rt groups were showed linerly increses, while, rts fed with the stndrd chow (the norml diet) showed higher feed intkes nd significnt difference (P <.5) compred to those fed with s without or with dioscorin interventions during 7-dy experiments (Figure 1B). Figure 1C showed the feed conversion rte of three rt groups during oesity-induced nd the whole experimentl stges which ws clculted from feed intkes (dt in the Figure 1B) divided y weight gined (dt in the Figure 1A). Rts fed with the stndrd chow (the norml diet) showed higher feed conversion rte nd significnt difference (P <.5) compred to those fed with s without or with dioscorin interventions during oesity-induced nd the whole experimentl stges (Figure 1C). It ment tht more of stndrd chow (the norml diet) were needed compred to those of to gin the sme weight. Figure 1D showed the orgn weights of three rt groups. Generlly, rts in the norml diet group showed significnt difference (P <.5) nd lower weights of dipose tissues (such s retroperitonel ft nd testicle ft) thn those in the HF diet groups. Other orgns except from kidney showed no significnt difference mong three groups (P >.5). Effects of dioscorin interventions on lood pressures of -induced oese rts Figure 2 showed the chnges of SBP (Figure 2A) nd DBP (Figure 2B) in three rt groups fter oesityinduced stges (dy 36 to dy 7). From the results of Figure 2A, the rts in the norml diet group showed lower SBP nd significnt differences (P <.5) compred to those in the group t 6-, 7-, 8-, nd 9- week. While, dioscorin intervention group ( + dio5) showed lower SBP nd significnt differences (P <.5) compred to those in the group t 7-, 8-, nd 9-week (corresponding to dioscorin intervention for 2 weeks, 3 weeks, nd 4 weeks, respectively) nd comprle to those in the norml diet group. From the results of Figure 2B, the rts in the norml diet group showed lower DBP nd significnt differences (P <.5) compred to those in the group t 6-, 7-, 8-, nd 9-week. Dioscorin intervention group ( + dio5) showed lowered SBP, however, did not exhiit significnt differences (P >.5) compred to those in the group t 7-, 8-, nd 9-week. These results showed tht the dioscorin intervention could improve lood pressure in oese rts, especil for SBP.

5 Shih et l. Botnicl Studies (215) 56:4 Pge 5 of 9 Distolic lood pressure (mmhg) Systolic lood pressure (mmhg) (week) (A) (B) + dio5 Norml diet Dioscorin (week) Figure 2 Effects of dioscorin interventions (5 mg/kg) on (A) systolic lood pressure nd (B) distolic lood pressures of high ft-diet induced oese rts. Blood pressure ws mesured t the end of ech week fter dioscorin intervention for 1-, 2-, 3-, nd 4-weeks y using n indirect til-cuff lood pressure meter (BP-98A, Softron Co. Ltd. Tokyo, Jpn). Dt were expressed s men ± SD. Multiple group comprisons under the sme treted time were performed using one-wy nlysis of vrince followed y the post hoc Tukey stest,nd vlues tht hve not een indicted with the sme lphet were significntly different (P <.5). HF, high-ft diet; ( + dio5), ym dioscorin (5 mg/kg, dio5) ws intervened dily concurrent. Effects of dioscorin interventions on impired glucose tolernces of -induced oese rts After fsting overnight, the impired glucose tolernce of rts in three groups were mesured y OGTT (Figure 3). Plsm glucose concentrtions (mg/dl) of the norml diet group in the, 5, 3, 6, 9, nd 12 min were ± 6.9, ± 18.67, ± 15.22, 13.9 ± 5.22, ± 7.57, nd ± 3.77, respectively; plsm glucose concentrtions (mg/dl) of group were 95.4 ± 5.66, ± 4.7, ± 5.4, ± 5.51, ± 7.2, nd 18.5 ± 8.34, respectively; plsm glucose concentrtions (mg/dl) of ( + dio5) group were ± 6.66, ± 3.6, 99.5 ± 8.42, ± 6.8, ± 7.52, nd ± 7.21, respectively. At the eginning, rts in the norml diet group nd ( + dio5) group showed lower plsm glucose concentrtions nd significnt differences (P <.5) compred to those in the group. Lter, rts in the ( + dio5) group kept the lowest plsm glucose concentrtions nd significnt differences (P <.5) mong three rt groups t ech 5-min, 3-min, Glucose (mg/dl) c + dio5 Norml diet Time (min) Figure 3 Effects of dioscorin interventions (5 mg/kg) on impired glucose intolernces of high ft-diet induced oese rts y orl glucose tolernce tests. Dt were expressed s men ± SEM. Multiple group comprisons under the sme treted time were performed using one-wy nlysis of vrince followed y the post hoc Tukey s test, nd vlues tht hve not een indicted with the sme lphet were significntly different (P <.5). HF, high-ft diet; ( + dio5), ym dioscorin (5 mg/kg, dio5) ws intervened dily concurrent. 6-min, 9-min, nd 12-min time intervls nd comprle to rts in the norml diet group t 9-min nd 12- min time intervls. The plsm glucose concentrtions of rts in the norml diet groups nd ( + dio5) group were then ck to the seline t 12 min, however, the plsm glucose concentrtions of rts in the group were still kept t higher levels (18.5 ± 8.34 mg/dl). From the OGTT dt, it ment tht the dioscorin intervention could improve impired glucose tolernces of induced oese rts. DPP IV inhiitory ctivities of peptic hydrolystes of ym dioscorin The reverse phse HPLC chromtogrms of peptic hydrolystes of ym dioscorin with moleculr mss etween 3 kd nd 1 kd (3 kd < peptide < 1 kd, Figure 4A) nd less thn 3 kd (peptide < 3kD, Figure 4B) were shown t Figure 4. These two hydrolytic frctions were used to nlyze the DPP IV inhiitory ctivities compred to the positive control of sitgliptin phosphte (Figure 4C). The sitgliptin phosphte showed dose-dependent inhiitions ginst DPP IV. Under 5 mg/ml doses, the peptide frctions of 3 kd < peptide < 1 kd nd peptide < 3kD, respectively, showed 8.82 ± 2.68 (%) nd ± 3.51 (%) DPP IV inhiitory ctivities which might e equivlent to sitgliptin phosphte of nm nd nm, respectively. Discussion The present niml experiments showed tht the dioscorin interventions t dose of 5 mg/kg dily for five c c c

6 Shih et l. Botnicl Studies (215) 56:4 Pge 6 of 9 (A) (B) DPP IV inhiition (%) (C) 3 D < peptide < 1 D peptide < 3 D Dioscorin hydrolystes Sitgliptin phosphte (nm) (mg/ml) Figure 4 DPP IV inhiitory ctivities of peptic hydrolystes of ym dioscorin. (A) HPLC chromtogrms of ym dioscorin peptic hydrolystes with moleculr mss etween 3 kd nd 1 kd, (B) HPLC chromtogrms of ym dioscorin peptic hydrolystes with moleculr mss less thn 3 kd, nd (C) DPP IV inhiitory ctivities of two frctions (5 mg/ml) of dioscorin peptic hydrolystes from moleculr cut-off memrne devices, nd sitgliptin phosphte ws cted s positive controls. weeks could improve SBP nd impired glucose tolernces, ut hd no effects on reductions of ody weight nd ft tissues, of 1-week -induced oese rts which the improved metolic risks were involved in MS criteri. The dioscorin nd its peptic hydrolystes t doses of 4 mg/kg were showed to hve ntihypertensive ctivities ginst spontneously hypertensive rts (SHR) (Lin et l. 26); the dioscorin t doses of 2 mg/kg or 8 mg/kg showed to lower oxidtive stress of BALB/c mice induced y sucutneous glctose injections (Hn et l. 214). The oesity my ply the centrl role in MS which the renin-ngiotensin system in dysfunctionl dipocytes will initite inflmmtion nd dyslipidemi, increse lood pressure nd decrese insulin sensitivity ccompnied with norml lood glucose (Frigolet et l. 213). Therefore, nti-oesity or prevention ginst oesity my reduce directly the risks of MS. There were severl reports concerning nti-oesity ctivity from nturl resources in rodent models induced y s, such s α-lipoic cid (Kim et l. 24), cryptotnshinone from Slvi militorrhiz (Kim et l. 27), rutin nd o- coumric cid (Hsu et l. 29), honokiol nd mgnolol (Kim et l. 213), pectin pentoligoscchride (Li et l. 213), high turine nd glycine contents of scllop protein (Tstesen et l. 214). The ove-mentioned models for -induced oese studies were generlly pplied

7 Shih et l. Botnicl Studies (215) 56:4 Pge 7 of 9 either y pretretment for period of time nd then tested smple interventions or co-tretment of HFdiet nd tested smples in the sme time. The vsorelxing peptides of Arg-Phe nd Ile-His-Arg-Phe derived from rice glutelin protein showed to lower food intkes in rodent models (Kgeyshi et l. 212; Kontni et l. 214) which mye hve nti-oesity ctivity. At present, it is not sure tht less mounts of dioscorin used with the similr MS preventive ctivities, or higher mounts of dioscorin used or co-tretments of HF-diet nd dioscorin used with nti-oesity ctivities. Regn-Shw et l. (27) suggested to use ody surfce re normliztion for dose trnsltion from niml to humn studies. It ws clculted tht the humn equivlent dose ws 8.11 mg/kg of humn ody weight from dioscorin intervention of 5 mg/kg of rt ody weight in the present experiment. An dult of 6 kg weigh might hve to consume out 49 mg dioscorin/dy to chieve enefits of improving MS nd need further investigtions. The ym dioscorin nd its peptic hydrolystes or synthesized peptides derived from dioscorin hd een reported to lower lood pressures using SHR s niml models (Lin et l. 26; Liu et l. 29,; Lin et l. 214). From the present results of Figure 2, dioscorin intervention t lest for two weeks showed to lower SBP of -induced oese rts nd comprle to the norml diet fed ones. The dysfunctionl dipocytes in oesity will increse circulting renin-ngiotensin systems, nd the generted ngiotensin II from ngiotensin I y ngiotensin converting enzyme (ACE) hydrolysis resulted in the higher lood pressures (Frigolet et l. 213). It ws reported tht oesity my elevte systemic oxidtive stress from overloded nutrients of s nd dvnced glyction endproduct genertions (Khn et l. 26). Glyction is the non-enzymtic modifiction of proteins through the reduction of sugrs nd their metolized intermedites, such s glyoxl or methylglyoxl, nd leds to the irreversile formtion of dvnced glyction end products (Kikuchi et l. 23). The hypertensive rt might increse oxidtive stress nd methylglyoxl mounts in vsculr smooth muscle cells (Wu nd Juurlink 22). The peptic hydrolystes of dioscorin showed ACE inhiitory ctivities (Hsu et l. 22) nd the synthesized peptides derived from pepsin hydrolystes in silico vsorelxing ctivities (Lin et l. 214). The synthesized peptides derived from pepsin hydrolystes in silico exhiited ntioxidnt nd ntiglyction ctivities in vitro nd in vivo (Hn et l. 213; Hn et l. 214,c). It ws proposed tht the dioscorin intervention showed to lower SBP of -induced oese rts might e from ACE inhiitory nd vsorelxing ctivities, nd in prt from ntioxidnt nd/or ntiglyction ctivities of ctive peptides fter dioscorin ingestions nd need further investigtions. From the results of Figure 3, it ws found tht the impired glucose tolernces in -induced oese rts were improved fter dioscorin interventions y OGTT methods. At the eginning nd the end of OGTT test, the oese rts exhiited significntly higher glucose levels compred to rts in norml diet group nd (HF + dio5) group. From the results of Figure 4, it ws found tht ym dioscorin peptic hydrolystes with moleculr mss 3 kd < peptide < 1 kd nd less thn 3 kd t doses of 5 mg/ml showed DPP IV inhiitory ctivities in vitro which might e equivlent to effects of nm nd nm of sitgliptin phosphte, respectively. Moreover, the undigested dioscorin ws lso used to evlute DPP IV inhiitory ctivity, under 5 mg/ml concentrtion, which showed 4.7% DPP IV inhiition (dt not show). The ntive dioscorin showed less nti-dpp IV ctivity compred to the 3 kd < peptide < 1 kd nd less thn 3 kd of dioscorin peptic hydrolystes. The results of DPP IV ctivity of the ntive nd digested dioscorin is in greement with the generl oservtion tht shortchin iologiclly ctive peptides cn e relesed nd sored in the smll intestine fter orl dministrtion (Pheln et l. 29) which my correlte with the improved OGTT. The sitgliptin phosphte, DPP IV inhiitor pproved y US FDA in 26, cn prolong GLP-1 iologicl ctivities to stimulte glucose-dependent insulin secretions nd for type 2 DM tretment in vivo (Idris nd Donnelly 27; Drucker et l. 27). The DPP IV inhiitor (vline-pyrrolidide) showed the improved glucose tolernce nd insulin secretion in -fed C57BL/6 J mice (Ahrén et l. 2). The mice fed (58% ft) together with DPP IV inhiitor (NVP DPP728) in the drinking wter for 8 weeks showed the improved glucose tolernce nd incresed circulting levels of insulin nd GLP-1 compred to only (Reimer et l. 22). It ws suggested tht the improved glucose tolernce in HF diet-induced oese rts fter dioscorin interventions might e in prt from DPP IV inhiitory ctivities of ctive peptides from dioscorin fter eing ingested nd need further investigtions. Conclusions In conclusion, ym dioscorin interventions exhiit the improved MS ctivities in oese rts nd peptic hydrolystes of ym dioscorin in vitro exhiit DPP IV inhiitory ctivities which the relted mechnisms my need further investigtions. Competing interests The uthors declre tht they hve no competing interests. Authors contriutions SLS, SYL, nd WCH prticipted the discussion nd concepts of experimentl designs, MS writing nd revision; YSL performed experiments. All uthors red nd pproved the finl mnuscript.

8 Shih et l. Botnicl Studies (215) 56:4 Pge 8 of 9 Acknowledgements The uthors would like to express thnks to Yun s Generl Hospitl, Kohsiung, Tiwn (13YGH-TMU-2-3) nd Ministry of Science nd Technology, Repulic of Chin (NSC B MY3) for finncil supports. Author detils 1 Yun s Generl Hospitl, Deprtment of Brest Surgery nd Cncer Center, Kohsiung, Tiwn. 2 Grdute Institute of Phrmcognosy, Tipei Medicl University, Tipei, Tiwn. 3 Deprtment of Primry Cre Medicine, Tipei Medicl University Hospitl, Tipei, Tiwn. 4 Deprtment of Generl Medicine, School of Medicine, Tipei Medicl University, Tipei, Tiwn. 5 Trditionl Herl Medicine Reserch Center, Tipei Medicl University Hospitl, Tipei, Tiwn. 6 Progrm for the Clinicl Drug Discovery from Botnicl Hers, Tipei Medicl University, Tipei, Tiwn. Received: 27 Octoer 214 Accepted: 7 Ferury 215 References Aguli MB, Mndrim-de-Lcerd CA (23) Hert nd lood pressure dpttions in Wistr rts fed with different high-ft diets for 18 months. Nutrition 19: Ahrén B, Holst JJ, Mårtensson H, Blkn B (2) Improved glucose tolernce nd insulin secretion y inhiition of dipeptidyl peptidse IV in mice. Eur J Phrmcol 44: Alerti KGMM, Eckel RH, Grundy SM, Zimmet PZ, Cleemn JI, Donto KA, Fruchrt J-C, Jmes WPT, Lori CM, Smith SC Jr (29) Hrmonizing the metolic syndrome: joint interim sttement of the interntionl dietes federtion tsk force on epidemiology nd prevention; ntionl hert, lung, nd lood institute; mericn hert ssocition; world hert federtion; interntionl therosclerosis society; nd interntionl ssocition for the study of oesity. Circultion 12: Andrikopoulos A, Blir AR, Deluc N, Fm BC, Proietto J (28) Evluting the glucose tolernce test in mice. Am J Physiol Endocrinol Met 295:E1323 E1332 Dniels J (26) Oesity: Americ s epidemic. Am J Nurs 16:4 49 Dy C (27) Metolic syndrome, or wht you will: definitions nd epidemiology. Dietes Vsc Dis Res 4:32 38 Drucker DJ (26) The iology of incretin hormones. Cell Met 3: Drucker D, Esley C, Kirkptrick P (27) Sitgliptin. Nt Rev Drug Discover 6:19 11 Frigolet ME, Nime Torres N, Armndo R, Tovr AR (213) The renin ngiotensin system in dipose tissue nd its metolic consequences during oesity. J Nutr Biochem 24: Hn CH, Liu JC, Fng SU, Hou WC (213) Antioxidnt ctivities of the synthesized thiol-contined peptides derived from computer-ided pepsin hydrolysis of ym tuer storge protein, dioscorin. Food Chem 138: Hn CH, Lin YS, Lin SY, Hou WC (214) Antioxidnt nd ntiglyction ctivities of the synthesised dipeptide, Asn-Trp, derived from computer-ided simultion of ym dioscorin hydrolysis nd its nlogue, Gln-Trp. Food Chem 147: Hn CH, Lin YF, Lin YS, Lee TL, Hung WJ, Lin SY, Hou WC (214) Effects of ym tuer protein, dioscorin, on ttenuting oxidtive sttus nd lerning dysfunction in D-glctose-induced BALB/c mice. Food Chem Toxicol 65: Hn CH, Lin YS, Lee TL, Ling HJ, Hou WC (214c) Asn-Trp dipeptides improve the oxidtive stress nd lerning dysfunctions in D -glctose-induced BALB/c mice. Food Funct 5: Htnk T, Inoue Y, Arim J, Kumgi Y, Usuki H, Kwkmi K, Kimur M, Mukihr T (212) Production of dipeptidyl peptidse IV inhiitory peptides from deftted rice rn. Food Chem 134: Hou WC, Chen HJ, Lin YH (2) Dioscorins from different Dioscore species ll exhiit oth cronic nhydrse nd trypsin inhiitor ctivities. Bot Bull Acd Sin 41: Hou WC, Lee MH, Chen HJ, Ling WL, Hn CH, Liu YW, Lin YH (21) Antioxidnt ctivities of dioscorin, the storge protein of ym (Dioscore tts Decne) tuer. J Agric Food Chem 49: Hsu FL, Lin YH, Lee MH, Lin CL, Hou WC (22) Both dioscorin, the tuer storge protein of ym (Dioscore lt cv. Tinong No. 1), nd its peptic hydrolystes exhiited ngiotensin converting enzyme inhiitory ctivities. J Agric Food Chem 5: Hsu CL, Wu CH, Hung SL, Yen GC (29) Phenolic compounds rutin nd o-coumric cid meliorte oesity induced y high-ft diet in rts. J Agric Food Chem 57: Idris I, Donnelly R (27) Dipeptidyl peptidse-iv inhiitors: mjor new clss of orl ntidietic drug. Dietes Oes Met 9: Ito M, Kondo Y, Nktni A, Hyshi K, Nruse A (21) Chrcteriztion of low dose streptozotocin-induced progressive dietes in mice. Envir Texicol Phrmcol 9:71 78 Kgeyshi T, Kontni N, Ymd Y, Mizushige T, Ari T, Kino K, Ohint K (212) Novel CCK-dependent vsorelxing dipeptide, Arg-Phe, decreses lood pressure nd food intke in rodents. Mol Nutr Food Res 56: Khn SE, Hull RL, Utzschneider KM (26) Mechnisms linking oesity to insulin resistnce nd type 2 dietes. Nture 444: Kikuchi S, Shinpo K, Tkeuchi M, Ymgishi S, Mkit Z, Sski N, Tshiro K (23) Glyction sweet tempter for neuronl deth. Brin Res Rev 41: Kim MS, Prk JY, Nmkoong C, Jng PG, Ryu JW, Song HS, Yun JY, Nmgoong IS, H J, Prk IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU (24) Anti-oesity effects of α-lipoic cid medited y suppression of hypothlmic AMP-ctivted protein kinse. Nt Med 1: Kim EJ, Jung SN, Son KH, Kim SR, H TY, Prk MG, Jo IG, Prk JG, Choe W, Kim SS, H J (27) Antidietes nd ntioesity effect of cryptotnshinone vi ctivtion of AMP-ctivted protein kinse. Mol Phrmcol 72:62 72 Kim YJ, Choi MS, Ch BY, Woo JT, Prk YB, Kim SR, Jung UJ (213) Long-term supplementtion of honokiol nd mgnolol meliortes ody ft ccumultion, insulin resistnce, nd dipose inflmmtion in high-ft fed mice. Mol Nut food Res 57: Koysi R, Akmine EH, Dvel AP, Rodrigues MAM, Crvlho CRO, Rossoni LV (21) Oxidtive stress nd inflmmtory meditors contriute to endothelil dysfunction in high-ft diet-induced oesity in mice. J Hypertens 28: Kontni N, Ome R, Kgeyshi T, Kneko K, Ymd Y, Mizushige T, Knmoto R, Ohint K (214) Chrcteriztion of Ile-His-Arg-Phe, novel rice-derived vsorelxing peptide with hypotensive nd norexigenic ctivities. Mol Nutr Food Res 58: Lcroix IME, Li-Chn ECY (212) Dipeptidyl peptidse-iv inhiitory ctivity of diry protein hydrolystes. Int Diry J 25:97 12 Lcroix IME, Li-Chn ECY (213) Inhiition of dipeptidyl peptidse (DPP)-IV nd α-glucosidse ctivities y pepsin-treted whey proteins. J Agric Food Chem 61: Li T, Zhu R, Dong Y, Liu Y, Li S, Chen G (213) Effects of pectin pentoligoscchride from hwthorn (Crtegus pinntifid Bunge. vr. Mjor) on the ctivity nd mrna levels of enzymes involved in ftty cid oxidtion in the liver of mice fed high-ft diet. J Agric Food Chem 61: Lin CL, Lin SY, Lin YH, Hou WC (26) Effects of tuer storge protein of ym (Dioscore lt cv. Tinong No. 1) nd its peptic hydrolyztes on spontneously hypertensive rts. J Sci Food Agric 86: Lin YS, Lu YL, Wng GJ, Ling HJ, Hou WC (214) Vsorelxing nd ntihypertensive ctivities of synthesized peptides derived from computer-ided simultion of pepsin hydrolysis of ym dioscorin. Bot Stud 55:49, doi:1.1186/s Liu YH, Ling HJ, Cheng HC, Liu YW, Hou WC (26) Comprisons of in vitro ntioxidnt ctivities of storge proteins in tuer of two Dioscore species. Bot Stud 47: Liu DZ, Ling HJ, Hn CH, Lin SY, Chen CT, Fn M, Hou WC (29) Feeding tril of instnt food contining lyophilised ym powder in hypertensive sujects. J Sci Food Agric 89: Liu YH, Lin YS, Liu DZ, Hn CH, Chen CT, Fn M, Hou WC (29) Effects of different types of ym (Dioscore lt) products on the lood pressure of spontneously hypertensive rts. Biosci Biotechnol Biochem 73: Lu YL, Chi CY, Liu YW, Hou WC (212) Biologicl ctivities nd pplictions of dioscorins, the mjor tuer storge proteins of ym. J Trdit Complement Med 2:41 46 Mentlein R (25) Therpeutic ssessment of glucgon-like peptide-1 gonists compred with dipeptidyl peptidse IV inhiitors s potentil ntidietic drugs. Expert Opin Investig Drugs 14:57 64 Pnchl SK, Brown L (211) Rodent models for metolic syndrome reserch. J Biomed Biotech 211:351982, doi: /211/ Pheln M, Aherne A, FitzGerld RJ, O'Brien NM (29) Csein-derived ioctive peptides: iologicl effects, industril uses, sfety spects nd regultory sttus. Int Diry J 19: Prentice AM (26) The emerging epidemic of oesity in developing countries. Int J Epidemiol 35:93 99 Regn-Shw S, Nihl M, Ahmd N (27) Dose trnsltion from niml to humn studies revisited. FASEB J 22: Reimer MK, Holst JJ, Ahrén B (22) Long-term inhiition of dipeptidyl peptidse IV improves glucose tolernce nd preserves islet function in mice. Eur J Endocrinol 146:

9 Shih et l. Botnicl Studies (215) 56:4 Pge 9 of 9 Rodgers RJ, Tschöp MH, Wilding JPH (212) Anti-oesity drugs: pst, present nd future. Dis Model Mech 5: Smyth S, Heron A (25) Dietes nd oesity: the twin epidemics. Nt Med 12:75 8 Tstesen HS, Keenn AH, Mdsen L, Kristinsen K, Liset B (214) Scllop protein with endogenous high turine nd glycine content prevents high-ft, high-sucrose-induced oesity nd improves plsm lipid profile in mle C57BL/6 J mice. Amino Acids 46: Woods SC, Seeley RJ, Rushing PA, D Alessio D, Tso P (23) A controlled high-ft diet induces n oese syndrome in rts. J Nutr 133: Wu L, Juurlink BHJ (22) Incresed methylglyoxl nd oxidtive stress in hypertensive rt vsculr smooth muscle cells. Hypertension 39: Zimmet P (1982) Type 2 (non-insulin-dependent) dietes n epidemiologicl overview. Dietologi 22: Sumit your mnuscript to journl nd enefit from: 7 Convenient online sumission 7 Rigorous peer review 7 Immedite puliction on cceptnce 7 Open ccess: rticles freely ville online 7 High visiility within the field 7 Retining the copyright to your rticle Sumit your next mnuscript t 7 springeropen.com