Effect of Senna alata (L) Roxb (Fabaceae) Leaf Extracts on alpha-amylase, alpha-glucosidase and Postprandial Hyperglycemia in Rats

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1 Tropil Journl of Phrmeutil Reserh Otoer 2015; 14(10): ISSN: (print); (eletroni) Phrmotherpy Group, Fulty of Phrmy, University of Benin, Benin City, Nigeri. All rights reserved. Originl Reserh Artile Aville online t Effet of Senn lt (L) Rox (Fee) Lef Extrts on lph-amylse, lph-gluosidse nd Postprndil Hyperglyemi in Rts Mutiu I Kzeem, Gniyt A Azeez nd Anofi OT Ashf* Phytomediine nd Phytophrmology Reserh Group, Deprtment of Plnt Sienes, University of the Free Stte, Qwqw Cmpus, Phuthditjh 9866, South Afri *For orrespondene: Emil: shfot@qw.ufs..z; Tel: ; Fx: Reeived: 17 July 2014 Revised epted: 22 July 2015 Astrt Purpose: To investigte the inhiitory effet of S. lt lef extrts on α-mylse nd α-gluosidse, nd its potentil for reduing postprndil lood gluose level of rts. Methods: The α-mylse nd α-gluosidse inhiitory potentils of etone, ethylette nd hexne extrts of S. lt were investigted y reting different onentrtions ( mg/ml) of the extrts with the enzymes nd sustrtes while the mode(s) of inhiition of oth enzymes were determined using the Linewever-Burk plot. The effet of orl dministrtion of the hexne extrt (with lowest IC 50 for α-gluosidse) of the plnt in surose-indued hyperglyemi rts ws lso determined y evluting fsting lood gluose levels. Results: The etone extrt of S. lt displyed the highest inhiitory tivity ginst α-mylse (IC 50 = 6.41 mg/ml) while hexne extrt exhiited highest inhiitory effet on α-gluosidse (IC 50 = 0.85 mg/ml). Both etone nd hexne extrts inhiited α-mylse nd α-gluosidse in ompetitive nd unompetitive mnner respetively. The hexne extrt of S. lt lso used signifint redution (p < 0.05) in the postprndil lood gluose level of surose-loded rts within two hours. Conlusions: It n e suggested tht one of the mehnisms of nti-dieti tion of S. lt lef is the inhiition of intestinl α-gluosidse. Keywords: Senn lt, Dietes, Postprndil hyperglyemi, Phytohemils, α-gluosidse, α- mylse Tropil Journl of Phrmeutil Reserh is indexed y Siene Cittion Index (SiSerh), Sopus, Interntionl Phrmeutil Astrt, Chemil Astrts, Emse, Index Copernius, EBSCO, Afrin Index Medius, JournlSeek, Journl Cittion Reports/Siene Edition, Diretory of Open Aess Journls (DOAJ), Afrin Journl Online, Bioline Interntionl, Open-J-Gte nd Phrmy Astrts INTRODUCTION Senn lt (Cssi lt) Linn. Rox (Leguminose), known s Ringworm plnt, winged senn, rw-rw plnt or king of the forest, is of gret importne in trditionl medil prtie in mny developing ountries [1]. In Nigeri, it is lled Asunwon oyino nd Nelkhi in the Western nd Estern regions of the ountry respetively. This plnt is indigenous to severl regions of the world suh s Afri nd South Ameri. It is n eret, tropil, nnul her of 0.15 m high with lethery, ompound yellowish-green leves [2]. It thrives in sunny nd moist res nd produes hrteristi offensive smell. Ethnophrmologil studies hve shown tht the leves of the plnt (S. lt) hve een used in the tretment of digestion-relted ilments suh s onstiption, dominl pin nd liver diseses [3]. It hs lso enjoyed wide usge in Trop J Phrm Res, Otoer 2015; 14(10): 1843

2 the mngement of dermtologi diseses like dermtitis, skin rsh, thlete s foot, ezem nd myosis [4]. The whole plnt is used in Brzil, Guteml nd Guine in the tretment of mlri, flu nd other infetious diseses [3]. Studies hve lso reported the ntiteril, ntifungl nd ntivirl properties of S. lt lef [1,6] s well s its lxtive [3] nd ortifient properties in wistr rts [2]. Of prtiulr interest is its use in the mngement of sugr-relted disorders suh s dietes mellitus nd hyperglyemi [5-7]. Although S. lt hs een vlidted sientifilly to possess nti-dieti property [6,7], there is no informtion on the possile mehnism y whih this plnt eliits its hypoglyemi effet. Therefore, the im of this study ws to determine the effet of lef extrts of S. lt on α-mylse nd α-gluosidse s well s its mode of inhiition of these enzymes, nd to ssess its effet on postprndil hyperglyemi. EXPERIMENTAL Smple olletion The leves of Senn lt were otined from Igndo Are of Lgos in Nigeri in My The plnt ws identified nd uthentited y the txonomist Dr AB Kdiri of the Deprtment of Botny, University of Lgos, Nigeri nd vouher speimen (LUH 5603) ws deposited in the University herrium. The leves were dried t 45 C in the oven, pulverized nd kept in ir-tight plsti gs. Experimentl nimls Mle wistr rts were otined from the Animl House of the University of the Free Stte, South Afri. They were housed in plsti ges under 12 h light/drk yle t C nd hd free ess to stndrd rt how nd tp wter d liitum. Ethil pprovl for the study ws otined from the Interfulty Ethis Committee of the University of the Free Stte, South Afri with pprovl numer NR 02/2013 nd ll experiments were performed ording to the Guide for the Cre nd Use of Lortory Animls [9]. Chemils nd regents Porine pnreti α-mylse, rt intestinl α- gluosidse nd pr nitrophenylgluopyrnoside (pnpg) were produts of Sigm-Adrih Co (St Louis, USA) while solule strh (extr pure) ws otined from JT Bker In. (Phillipsurg, USA). Other hemils nd regents were of nlytil grde nd wter used ws glss-distilled. Preprtion of plnt extrts The powdered leves were divided into three portions of 20 g eh nd these were extrted with etone, ethylette or hexne. They were ll left to steep in overed Erlenmeyer flsk for 24 h nd the resulting infusions were dented, filtered with Whtmn 150 mm filter pper nd evported to dryness in rotry evportor (Cole Prmer SB 1100, Shngi, Chin). α-amylse inhiitory ssy This ssy ws rried out using modified proedure of MCue nd Shetty [10]. The extrt (250 μl, mg/ml) ws pled in test tue nd 250 μl of 0.02 M sodium phosphte uffer (ph 6.9) ontining α-mylse solution (0.5 mg/ml) ws dded. This solution ws preinuted t 25 C for 10 min, fter whih 250 μl of 1 % strh solution in 0.02 M sodium phosphte uffer (ph 6.9) ws dded t timed intervls nd then inuted t 25 C for 10 min. The retion ws terminted y dding 500 μl of dinitrosliyli id (DNS) regent. The tues were then inuted in oiling wter for 5 min nd ooled to room temperture nd the retion mixture ws diluted with 5 ml distilled wter. Asorne then ws mesured t 540 nm in spetrophotometer (Spetruml S23A, Gloe Medil Englnd). Distilled wter nd rose were used s ontrol nd stndrd respetively. The α-mylse inhiitory tivity ws lulted s in Eq 1. Inhiition (%) = {(A-Ae)/A}100.. (1) where A nd Ae re the sorne vlue of the ontrol nd extrt respetively. Conentrtions of extrts resulting in 50 % inhiition of enzyme tivity (IC 50 ) were determined grphilly using Mirosoft Exel Mode of α-mylse inhiition The mode of inhiition of α-mylse y the lef extrt ws onduted using the most potent extrt ording to the modified method desried y Ali et l [11]. Briefly, 250 μl of the extrt (5 mg/ml) ws pre-inuted with 250 μl of α-mylse solution (0.5 mg/ml) for 10 min t 25 C in one set of tues. In nother set of tues, α-mylse ws pre-inuted with 250 μl of 0.02 Trop J Phrm Res, Otoer 2015; 14(10): 1844

3 M sodium phosphte uffer (ph 6.9). Strh solutions (250 μl) t inresing onentrtions ( mg/ml) were dded to oth sets of retion mixtures to strt the retion. Eh mixture ws then inuted for 10 min t 25 C, nd then oiled for 5 min in wter th (TW20, Julo GmH, Germny) fter ddition of 500 μl of DNS to stop the retion. The mount of reduing sugrs relesed ws determined olorimetrilly using mltose stndrd urve nd onverted to retion veloities (mm/min). A doule reiprol plot (Linewever-Burk) (1/v ginst 1/[S]) where v is retion veloity nd [S] is sustrte onentrtion ws plotted to determine the mode of inhiition. α-gluosidse inhiitory ssy The effet of the plnt extrts on α-gluosidse tivity ws determined ording to the method desried y Kim et l [12]. The sustrte solution (3.0 mm), p-nitropheynyl gluopyrnoside (pnpg) ws prepred in 20 mm phosphte uffer, ph μl of α- gluosidse (E.C ) (0.5 mg/ml) ws preinuted with 50 μl of the different onentrtions of the extrts for 10 min. Then 50 μl of 3.0 mm pnpg dissolved in 20 mm phosphte uffer (ph 6.9) ws dded to strt the retion. The retion mixture ws inuted t 37 C for 20 min nd stopped y dding 2 ml of 0.1 M N 2 CO 3 nd the α-gluosidse tivity ws determined y mesuring the yellow oloured pr-nitrophenol relesed from pnpg t 405 nm using spetrophotometer (Spetruml S23A, Gloe Medil Englnd). Distilled wter nd rose were used s ontrol nd stndrd respetively. Perentge inhiition ws lulted s in Eq 2. Inhiition (%) = {(A-Ae)/A}100. (2) where A nd Ae re the sornes of the ontrol nd extrt respetively. Conentrtions of extrts resulting in 50 % inhiition of enzyme tivity (IC 50 ) were determined grphilly using Mirosoft Exel. Mode of α-gluosidse inhiition The mode of inhiition of α-gluosidse y the extrts ws determined using the extrt with the lowest IC 50 ording to the modified method desried y Ali et l [11]. Briefly, 50 μl of the 5 mg/ml extrt ws pre-inuted with 100 μl of α-gluosidse solution (0.5 mg/ml) for 10 min t 25 C in one set of tues. In nother set of tues, α-gluosidse ws pre-inuted with 50 μl of phosphte uffer (ph 6.9). At inresing onentrtions ( mg/ml), 50 μl of pnpg ws dded to oth sets of retion mixtures to strt the retion. The mixture ws inuted for 10 min t 25 C nd 500 μl of N 2 CO 3 ws dded to stop the retion nd sorne ws red t 405 nm using spetrophotometer (Spetruml S23A, Gloe Medil Englnd). The mount of prnitrophenol relesed ws determined olorimetrilly using pr-nitrophenol lirtion urve nd onverted to retion veloities. A doule reiprol (Linewever-Burk) plot (1/v ginst 1/[S]) where v is retion veloity nd [S] is sustrte onentrtion ws plotted to determine the mode of inhiition. Surose-tolerne test Ten norml rts were divided into two groups of five rts (n = 5) eh: Group A ws designted s the ontrol rts while Group B ws the hexne-extrt treted rts. After n overnight fst (18 h), groups B rts were dministered hexne extrt of S. lt lef (250 mg/kg ody weight) orlly y mens of n orogstri tue while the rts in group A reeived distilled wter (vehile). Both groups were orlly dministered 3 g/kg ody weight of surose solution 30 min fter the dministrtion of the extrt. Postprndil lood gluose levels were then mesured efore (0 min) nd t 30, 60, 90 nd 120 min fter orl dministrtion of surose using Gluometer 4 Ames (Byer Dignostis, Germny). The postprndil lood gluose levels were plotted nd ompred with those of ontrol rts. Sttistil nlysis Sttistil nlysis ws performed using GrphPd Prism 5 sttistil pkge (GrphPd Softwre, USA). The dt were nlysed y one wy nlysis of vrine (ANOVA) followed y Bonferroni test. All results were expressed s men ± SEM (n = 3). RESULTS Figure 1() shows the perentge inhiition of α- mylse tivity y different extrts of S. lt. At ll onentrtions tested, the vlues otined for the three extrts were signifintly different (p < 0.05) from one nother exept t 10 mg/ml where the vlues for ethylette nd hexne extrt were similr. Aetone extrt exhiited the highest perentge inhiition of the enzyme when ompred to other extrts. The IC 50 vlues generted from the perentge inhiition revels tht of ll the extrts, etone extrt of S. lt hs the lowest IC50 vlue (6.41 mg/ml) Trop J Phrm Res, Otoer 2015; 14(10): 1845

4 ut ws higher thn tht of rose (5.19 mg/ml) (Tle 1). Kineti nlysis of the α- mylse inhiition y the etone extrt of S. lt lef using Linewever-Burk plot shows tht it displyed ompetitive mode of inhiition (Figure 1). The perentge inhiition of α-gluosidse tivity y etone, ethylette nd hexne extrts of S. lt re presented in Figure 2(). At lower onentrtions ( mg/ml), the vlues for hexne extrt were signifintly higher (p < 0.05) thn the vlues for other % Inhiition 60 Aetone Ethylette Hexne () Conentrtion (mg/ml) extrts. There were signifint differenes (p < 0.05) mong ll the extrts t 5 mg/ml ut t the highest onentrtion (10 mg/ml), the vlues otined for ll the extrts were not signifintly different from one nother. The IC 50 vlues generted from the perentge inhiition of the extrts revels tht hexne extrt hs the lowest vlue (0.85 mg/ml) ompred to other extrts nd rose (Tle 1). Kineti nlysis of the α-gluosidse inhiition y the hexne extrt of S. lt lef using Linewever-Burk plot shows tht it displyed n unompetitive mode of inhiition (Figure 2). Extrt Control 1/v (mm/min) () /[s] (mm) -1 Figure 1: Inhiitory poteny of S. lt lef extrts ginst α-mylse tivity (). Mode of inhiition of α- mylse y etone extrt of Senn lt (). Vlues re expressed s men ± SEM of triplite determintions. Mens not shring ommon letter t the sme onentrtion re signifintly different (p < 0.05) Tle 1: IC 50 vlues for the inhiition of α-mylse nd α-gluosidse y different extrts of S. lt lef nd rose Extrts IC 50 (mg/ml) α-amylse α-gluosidse Arose 5.19 ± ± 0.02 Aetone 6.41 ± ± 0.10 Ethylette ± ± 0.07 Hexne ± ± 0.02 The vlues re expressed s mens ± SEM of triplite determintions. Mens down vertil olumn not shring ommon letter re signifintly different (P < 0.05). Arose is the stndrd α-mylse nd α-gluosidse inhiitor % Inhiition () 100 Aetone Ethylette 80 Hexne Extrt Control 1/v (mm/min) () Conentrtion (mg/ml) /[s] (mm) -1 Figure 2: Inhiitory poteny of S. lt lef extrts ginst α-gluosidse tivity (), Mode of inhiition of α- gluosidse y hexne extrt of Senn lt (). Vlues re expressed s mens ± SEM of triplite determintions. Mens not shring ommon letter t the sme onentrtion re signifintly different (p < 0.05) Trop J Phrm Res, Otoer 2015; 14(10): 1846

5 The effet of orl dministrtion of the hexne extrt of S. lt on surose-indued hyperglyemi is shown in Figure 3. The result shows tht the rise in postprndil lood gluose level of the ontrol nd extrt-treted rts were similr t the initil period fter the dministrtion of extrt, until t 60 min when the extrt signifintly redued (p < 0.05) the gluose level. This trend of redution in the extrt-treted hyperglyemi nimls ontinued ut not signifint y the end of the experiment. This is lso orroorted y the signifint differene etween the re under urve (AUC) of the ontrol nd tht of the hexne extrt-treted nimls (Tle 2). DISCUSSION Dietes mellitus is metoli disorder hrterized y hyperglyemi resulting from defets in insulin seretion, insulin tion or oth [13]. It ffets the metolism of rohydrtes, fts nd proteins in the ody, leding to severl omplitions suh s nephropthy, neuropthy nd retinopthy [14]. Aording to the Interntionl Dietes Federtion, the numer of people living with dietes in 2013 ws 382 million worldwide nd is projeted to rise to 592 million y 2035 [15]. The mngement of hyperglyemi is the hllmrk of tretment in dietes. One of the therpeuti pprohes for deresing postprndil hyperglyemi is to retrd the digestion nd sorption of rohydrtes. This n e hieved y the inhiition of rohydrte hydrolyzing enzymes, α-mylse nd α- gluosidse, in the digestive trt [16]. Though syntheti α-gluosidse inhiitors suh s rose nd vogliose re presently in use, their use is edevilled y undesirle side effets suh s nuse, dirrhoe nd liver filure [17]. In this study, the low perentge inhiition of α- mylse y the extrts of Senn lt is pointer to the ft tht the plnt is mild inhiitor of the enzyme. This is desirle of ntidieti mediinl plnts in order to prevent some of the side effets produed y syntheti drugs [18]. The hrteristi ompetitive inhiition of α- mylse exhiited y the etone extrt of S. lt suggests tht the tive omponent in the extrt is struturlly similr to the sustrte. This suggests tht the extrt inds reversily to the tive site of the enzyme nd oupies it in mutully exlusive mnner with the sustrte [19]. Blood Gluose (mg/dl) 150 Control Extrt Time (Min) Figure 3: Effet of dministrtion of hexne extrt of S. lt on lood gluose level of surose-loded norml rts. * Vlues re signifintly lower thn the ontrol. * Tle 2: Phrmokineti prmeters of ontrol nd S. lt treted rts fter surose ingestion PK Prmeters Groups Control Extrt C mx (mg/dl) ± ± 3.10 T mx (min) ± ± 0.00 AUC (mg/dl.min) ± ± 16.70* The vlues re expressed s mens ± SEM of triplite determintions. *Men vlues re signifintly different from the ontrol. (C mx is the mximum lood gluose level of the rts, T mx is the time t whih the mximum lood gluose level ws ttined nd AUC is the re under the urve) The strong inhiition of α-gluosidse y hexne extrt of S. lt s depited y the lowest IC 50 (0.85 mg/ml) suggests tht it ontins potent α- gluosidse inhiitor, inditing potentil role s n ntidieti nturl soure. This is euse ny plnt whih is strong inhiitor of α-gluosidse ould serve s effetive therpy for postprndil hyperglyemi with miniml side effets [20]. Linewever-Burk plot showed tht the hexne extrt of this plnt inhiited α- gluosidse in n unompetitive mnner. This suggests tht the inhiitor hs no struturl similrity with the sustrte nd the inhiitor inds exlusively to the enzyme-sustrte omplex yielding n intive enzyme-sustrteinhiitor omplex [21]. Trop J Phrm Res, Otoer 2015; 14(10): 1847

6 In order to onfirm the ntihyperglyemi effet of the hexne extrt of S. lt in-vivo, the effet of the orl dministrtion of hexne extrt of S. lt on postprndil hyperglyemi ws tested. The redution in the lood gluose level of the S. lt extrt-treted rts ompred to the ontrol nimls suggest tht the hexne extrt of the plnt slowed down the digestion nd sorption of surose in the nimls. CONCLUSION Findings from this study show tht of ll the S. lt extrts tested, etone nd hexne extrt provided the most effetive inhiition of α- mylse nd α-gluosidse, respetively, nd tht their mode of inhiiting oth enzymes is ompetitive nd unompetitive inhiition, respetively. The hexne extrt lso redues surose-indued postprndil hyperglyemi in rts. Therefore, it n e onluded tht S. lt lef extrts inhiit α-mylse s well s α- gluosidse nd redued postprndil hyperglyemi in rts. ACKNOWLEDGEMENT The uthors herey knowledge the Ntionl Reserh Foundtion (NRF), South Afri for the NRF Freestnding Postdotorl Fellowship grnted Dr MI Kzeem tenle t the Phytomediine nd Phytophrmology Reserh Unit of the Deprtment of Plnt Sienes, University of the Free Stte, Qwqw Cmpus, Phuthditjh, South Afri. REFERENCES 1. Adedyo O, Anderson WA, Moo-Young M, Sniekus V, Ptil PA, Kolwole DO. Phytohemistry nd ntiteril tivity of Senn lt flower. Phrm Biol 2001; 39 (6): Ykuu MT, Adeshin AO, Oldiji AT, Aknji MA, Oloyede OB, Jimoh GA, Oltinwo AWO, Afolyn AJ. Aortifient potentil of queous extrt of Senn lt leves in rts. J Reprod Contrep 2010; 21(3): Henneelle T, Weniger B, Joseph H, Shpz S, Billeul F. Senn lt. Fitoterpi 2009; 80: Ajiesin KK, Ekpo BA, Bl DN, Essien EE, Adesny SA. Ethnootnil survey of Akw Iom Stte of Nigeri. J Ethnophrmol 2008; 115: Ao KA, Fred-Jiyesimi AA, Jiyesimi AEA. Ethnootnil studies of mediinl plnts used in the mngement of dietes mellitus in South Western Nigeri. J Ethnophrmol 2008; 115: Wordulypinij W, Soonthornhreonnon N, Wiwt C. In vitro HIV type 1 reverse trnsriptse inhiitory tivities of Thi mediinl plnts nd Cnn indi L. rhizomes. J Ethnophrmol 2005; 101: Plnihmy S, Ngrjn S, Devsgym M. Effet of Cssi lt lef extrt on hyperglyemi rts. J Ethnophrmol 1988; 22: Villsenor IM, Cnls AP, Psu MPI, Sndo MN, Soliven LAP. Biotivity studies on Cssi lt Linn. lef extrts. Phytother Res 2002; 16, S93 S Ntionl Ademy of Sienes. Guide for the re nd use of lortory nimls, 8th Ed., Wshington, USA, Ntionl Ademi Press, MCue PP, Shetty K. Inhiitory effets of rosmrini id extrts on porine pnreti α-mylse in vitro. Asi P J Clin Nutr 2004; 13: Ali H, Houghton PJ, Soumynth A. Alph-mylse inhiitory tivity of some Mlysin plnts used to tret dietes with prtiulr referene to Phyllnthus mrus. J Ethnophrmol 2006; 107: Kim YM, Jeong YK, Wng MH, Lee WY, Rhee HI. Inhiitory effets of pine rk extrt on lphgluosidse tivity nd postprndil hyperglyemi. Nutrition 2005; 21: Khn A, Zmn G, Anderson RA. By leves improve gluose nd lipid profile of people with Type 2 dietes. J Clin Biohem Nutr 2009; 44: Afolyn AJ, Sunmonu TO. In vivo studies on ntidieti plnts used in South Afrin Herl Mediine. J Clin Biohem Nutr 2010; 47: Interntionl Dietes Federtion (IDF). IDF Dietes Atls, 6th ed., Brussels, Belgium, Mtsui T, Ogunwnde IA, Aesundr KJM, Mtsumoto K. Anti-hyperglyemi potentil of nturl produts. Mini Rev Med Chem 2006; 6 (3): Irhim MA, Islm MS. Butnol frtion of Khy seneglensis root modultes β-ell funtion nd meliortes dietes-relted iohemil prmeters in type 2 dietes rt model. J Ethnophrmol 2014; 154: Dong H, Li M, Zhu F, Liu F, Hung J. Inhiitory potentil of trilotin from Lithorpus polysthyus Rehd ginst α-gluosidse nd α-mylse linked to type 2 dietes. Food Chem 2012; 130: Aesundr KJM, Mtsui T, Mtsumoto K. α-gluosidse inhiitory tivity of some Sri Lnk plnt extrts, one of whih, Cssi uriult, exerts strong ntihyperglyemi effet in rts omprle to the therpeuti drug rose. J Agri Food Chem 2004; 52: Kwon YI, Apostolidis E, Shetty K. In vitro studies of eggplnt (Solnum melongen) phenolis s inhiitors of key enzymes relevnt for type 2 dietes nd hypertension. Bioresoure Tehnol. 2008; 99: King JB, West MB, Cook PF, Hnign MH. A Novel, Speies-speifi lss of unompetitive inhiitors of γ- Glutmyl trnspeptidse. J Biol Chem 2009; 284(14): Trop J Phrm Res, Otoer 2015; 14(10): 1848