Hypoglycaemic effects of the aqueous seed extract of Hunteria umbellata in normoglycaemic and glucoseand nicotine-induced hyperglycaemic rats

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1 Interntionl Journl of Applied Reserch in Nturl Products Vol. 2(1), pp. 9-18, Mrch-April 2009 Avilble online Helthy Synergies Publictions Originl Article Hypoglycemic effects of the queous seed extrct of Hunteri umbellt in normoglycemic nd glucosend nicotine-induced hyperglycemic rts Adeneye AA b, Adeyemi OO b, * Deprtment of Phrmcology, Fculty of Bsic Medicl Sciences, Lgos Stte University College of Medicine, Ikej, Lgos Stte, Nigeri b Deprtment of Phrmcology, College of Medicine, University of Lgos, Idi-Arb, Lgos Stte, Nigeri Summry: Dry seeds of the plnt, Hunteri umbellt K. Schum (fmily: Apocyncee), re highly vlued in Africn trditionl medicine in the tretment of vrious humn diseses, including dibetes mellitus nd obesity. In the present study, the hypoglycemic nd weight loss effects of mg/kg of the queous seed extrct of Hunteri umbellt (HU) were investigted in norml nd drug-induced hyperglycemic rts. In ddition, the cute orl toxicity using the preliminry nd the min tests of the Up-nd-Down Procedure ccording to OECD/OCDE Test Guidelines on Acute Orl Toxicity ws conducted. Phytochemicl nlysis of the queous seed extrct ws lso crried out. Results showed tht HU cused progressive nd significnt (p<0.05, p<0.01 nd p<0.001) dose-relted reduction in the blood glucose concentrtions in norml nd drug-induced hyperglycemic rts, n effect, which ws more thn tht of glibenclmide nd medited vi inhibition of intestinl glucose uptke nd drenergic homeosttic mechnisms. HU lso cused significnt (p<0.05 nd p<0.01) dose-dependent reduction in the verge body weight of treted rts when compred to untreted rts. The cute orl toxicity study showed tht the plnt extrct hd n LD50 of 1020 mg/kg nd s such slightly toxic. Results of the phytochemicl nlysis of HU reveled the presence of lkloids, flvonoids, tnnins nd glycosides. Thus, the dt generted in the present study hs strong positive correltion with its folkloric use in the tretment of suspected type 2 dibetic ptients, lthough its use should be with gret cution. Industril relevnce: The dt generted in this study suggest tht HU could be very useful in the mngement of type 2 dibetes mellitus lthough severl further studies will still be required t identifying the ctive hypoglycemic phytocomponent(s) nd their possible mechnisms of ction. Keywords: Hunteri umbellt (K. Schum), queous seed extrct, glucose- nd nicotine-induced hyperglycemic rts, hypoglycemi, cute orl toxicity, phytochemicl nlysis Introduction Dibetes mellitus (DM) remins one of the mjor chronic disorders of crbohydrte, lipid, nd protein metbolism chrcterized by persistent elevtions of fsting blood glucose equl or greter thn 140 mg/dl tken on t lest two seprte occsions, resulting from prtil or complete cesstion of insulin synthesis or secretion nd/or peripherl resistnce to insulin ction (Murry nd Pizzorno, 1997). DM is often ssocited with incresed risk of pnsystemic complictions which include ischemic hert disese, nephropthy, retinopthy, neuropthy, ulcertion nd gngrene of extremities (Murry nd Pizzorno, 1997; Rotshteyn nd Zito, 2004). DM could occur lone but more often co-exists with other systemic diseses such s hypertension, dyslipidemi, ischemic hert disese, renl diseses etc. As result, DM nd its ssocited complictions hve significnt impct on helth, qulity of life nd life expectncy of its sufferers. In 1998, World Helth Orgniztion estimted tht pproximtely 120 to 140 million people were globlly ffected by DM (WHO, 1999). In 2000, this figure incresed to more thn 177 millions (Kld et l., 2008) nd is projected to increse to 221 millions by 2010 (Amos et l., 1997) nd to double by the yer 2025 (King et l., 1998; Kld et l., 2008). Also, *Corresponding Author: Tel: E-mil: oodey@yhoo.com 9

2 Hypoglycemic effects of the queous seed extrct of Hunteri umbellt epidemiologicl studies showed tht the globl epidemic of DM is worse or greter in developing thn the developed countries (Oput, 2002). In response to this globl helth chllenge, the WHO Expert Committee on dibetes mellitus recommended n urgent nd further evlution of the folkloric methods of mnging the disese (WHO, 1980; WHO, 2002) becuse of high mortlity nd morbidity rising from its ttendnt complictions nd limittions ssocited with the use of conventionl ntidibetic drugs (Adeneye et l., 2006). The limittions include inccessibility to helth fcilities where the drugs re vilble, high cost, nd rigid tretment regimen (Adeneye nd Agbje, 2008). In response to this recommendtion, severl medicinl plnts re currently being investigted for their hypoglycemic efficcies. One of the severl indigenous plnts used in the locl tretment of DM mong Yorub (South-West Nigeri) is the dry seeds of Hunteri umbellt. Hunteri umbellt (K. Schum) Hllier which is synonymous with Picrlim umbellt K. Schum, nd Polydo umbellt (K. Schum) Stpf., belongs to the fmily Apocyncee. The plnt is smll glbrous tree mesuring 2-5 feet in girth nd feet high which grows well nd ubiquitous to the tropicl West Africn forest groove (Sofowor, 1982). Its leves re green in colour, brodly elliptic to oblong in shpe mesuring cm long, 5-9 cm brod with pirs of lterl veins (Hutchinson nd Dlziel, 1937). The xis nd brnches of its white, well-scented inflorescence re mrkedly thickened t their pex (Hutchinson nd Dlziel, 1937). The plnt is loclly known s Abeere mong the Yorub (South-West Nigeri). In Africn folk medicine, vrious prts (especilly the leves, roots nd brks) of Hunteri umbellt plnt re highly vlued for the tretment of vrious veterinry nd humn diseses. For exmple, decoction mde from the plnt stem nd root is reputed for its ntihelminthic ctivities nd in the tretment of swellings (Gill, 1992). The plnt leves nd pulp re eqully used by West Africn trditionl midwives to tret pregnncy relted ilments nd to induce or ugment lbour t term (Flodun et l., 2006). Recently, the dose dependent nd the mechnism of ction of the contrctile effects of the queous pulp extrct of the plnt in isolted non-pregnnt uterus were reported (Flodun et l., 2006). Recent ethnobotnicl survey done by us reveled tht mong the Yorub herblists (South-West Nigeri), pulverized dry seeds of Hunteri umbellt re eqully reputed nd highly vlued for the tretment of fever, pin, bdominl colic nd discomforts, dibetes mellitus nd obesity in its suspected sufferers. In the locl tretment of dibetes mellitus, n verge of 2-3 tespoonfuls (10-15 g) of the pulverized plnt seed soked in glss full of hot wter for min is often recommended for n dult to be tken orlly per dy. This trnsltion nd the result of our preliminry study in rt model informed our decision to choose the dose rnge of mg/kg/dy of the extrct. However, despite the ncestrl use of the plnt decoction in the tretment of suspected dibetic ptients, there is derth in scientific vlidtion of this therpeutic use. The present study is, therefore, designed to investigte the effects of cute nd repeted grded orl doses ( mg/kg/dy) of the queous seed extrct of Hunteri umbellt in norml rts for 14 dys, nd glucose- nd nicotine-induced hyperglycemic rts for 6 hours. In ddition, the cute orl toxicity s well s the phytochemicl nlysis of the extrct ws conducted. Mterils nd Methods Collection of Plnt Mterils Fresh leves, inflorescence nd mture pods of the Hunteri umbellt plnt were collected from the deciduous forest of Odorsnyin District of Ijebu-Igbo in Ijebu North Locl Government Are of Ogun Stte, Nigeri, in the month of April, Botnicl identifiction ws done by Mr. T. K. Odewo, Chief Superintendent Officer, Txonomy Section, Forest Reserch Institute of Nigeri, Ibdn, Oyo Stte, Nigeri. Voucher specimen ws deposited in the institution herbrium with the reference no. FHI , llotted. Plnt uthentiction ws done by Dr. A.B. Kdiri, The Herbrium, Botny nd Microbiology Deprtment, the University of Lgos, Akok, Lgos Stte, Nigeri. Hunteri umbellt seeds were hrvested from the collected fresh fruit pods. The seeds were gently wshed in tp wter nd completely dried under room temperture (30 ± 2 ºC) for 4 weeks protected from direct het or sunlight. When dried, the seeds were de-coted of their lightbrown thin cotings. Preprtion of the queous seed extrct of Hunteri umbellt 100 g of the dry seeds ws pulverized to white-to-light brown fine powder using domestic blender. 25 g of the fine powdered smple ws boiled in 500 ml of distilled wter in 1 L Pyrex beker for 1 hr under continuous stirring. The homogente ws llowed to cool for bout 6 hr before it ws rpidly filtered through piece of clen white cloth. The filtrte ws then trnsferred to n erted oven preset t 40 ºC nd completely dried until deep brown, romtic solid residue ws obtined. The yield obtined ws 45% (w/w). The residue, thus obtined, ws stored in ir- nd moisture-tight continer which ws kept in refrigertor mintined t -4 ºC. From this, fresh stock ws reconstituted in distilled wter t concentrtion of 100 mg/ml, whenever needed. Experimentl Animl Twenty femle Wistr rts, weeks nd weighing between g were employed for cute orl toxicity study using the Up nd Down Procedure ccording to OECD/OCDE Test Guidelines on Acute Orl 10

3 Adeneye nd Adeyemi Toxicity under computer-guided sttisticl progrmme - AOT425sttPgm, version: 1.0. (2001). In ddition, totl of one hundred nd two weeks old, mle Wistr rts, weighing g, were used for the cute nd repeted dose experiments, respectively. The rts were obtined from the Animl House of the Lgos Stte University College of Medicine, Ikej, Lgos Stte, in the month of September, The rts were hndled in ccordnce with interntionl principles guiding the Use nd Hndling of experimentl nimls (United Sttes Ntionl Institutes for Helth, 1985). The rts were mintined on stndrd rt feed (Livestock Feeds, Ikej, Lgos Stte) nd wter which were mde vilble d libitum. The rts were mintined t n mbient temperture between ºC, humidity of 55 ± 5%, nd stndrd (nturl) photoperiod of pproximtely 12 hr of light (06:30 hr 18:30 hr) lternting with pproximtely 12 hr of drkness (18:30 hr - 06:30 hr). Acute orl toxicity studies of Hunteri umbellt queous seed extrct using limit dose test nd the min test of Up nd Down Procedure An initil preliminry orienttion-test of cute orl toxicity study ws conducted using the limit dose test of Up nd Down Procedure ccording to OECD/OCDE Test Guidelines on Acute Orl Toxicity under computerguided sttisticl progrmme - AOT425sttPgm, version: 1.0., t limit dose of 2000 mg/kg body weight/orl route nd defult t n ssumed sigm of 0.5 mg/kg/orl, using the method dopted by Adeneye et l. (2006b). Due to lethlity of the two sequentilly treted rts t 2000 mg/kg/orl route of HU, the min test of the Up nd Down Procedure ws subsequently conducted, using the dose progression of 175 mg/kg/orl, 550 mg/kg/orl nd 2000 mg/kg/orl of HU. The procedure for the min test ws conducted in strict complince with the guidelines of the min test of Up nd Down Procedure ccording to OECD/OCDE Test Guidelines on Acute Orl Toxicity under computer-guided sttisticl progrmme - AOT425sttPgm (AOT, 2001). Preliminry Phytochemicl nlysis The presence of sponins, tnnins, lkloids, flvonoids, nthrquinones, glycosides nd reducing sugrs were determined by the simple nd stndrd qulittive nd quntittive methods described by Trese nd Evns (1989) nd Sofowor (1993). The simple quntittive nlysis of the extrct ws bsed on the intensity of the colour chnge. Briefly described, the qulittive phytochemicl nlysis of the crude seed powder of Hunteri umbellt ws determined s follows: Tnnins: 200 mg of the plnt mteril ws dissolved in 10 ml of distilled wter nd then filtered. A 2 ml of filterte ws pipetted into test tube fter which 2 ml of 15% FeCl 3 ws dded. Colour chnge ws observed. Blue-blck presence indicted the presence of tnnins. Alkloids: 200 mg of the plnt mteril ws extrcted with 200 ml of methnol for 20 minutes on wter bth nd then filtered. To 2 ml of the cold wter extrct in different tubes, ws dded 6 drops of different lkloids regents, nmely: Drgendorff s or Myer s or Wgners s regent. Presence nd colours of ny precipitte were noted. Cremish precipitte or brownish-red precipitte or ornge precipitte indicted the presence of respective lkloids. Cynogenic glycosides: 200 mg of powdered plnt mteril ws plced in ech of 3 different test tubes lbeled A, B nd C, respectively. The powder in test tubes A nd B were moistened with 5 ml of wter, while tht in test tube C ws left dry. 3 pieces of freshly prepred sodium picrte pper were inserted into the mouth of ech tube nd stoppered. Test tube B ws plced in wter bth while test tube A nd C were kept t room temperture. After 30 minutes the colour of the picrte ppers in ech of the test tube were observed nd recorded. Crdic glycosides: - Kedde s test for lctone ring in crdic glycosides: 5 g of plnt mteril ws boiled with 50 ml of wter to obtin wter extrct of the plnt. The extrct ws concentrted to dryness nd re-dissolved in 10 ml of methnol. To 2 ml of this, 1 ml of solution of 2% of 3, 5-dinitrobenzoic cid in methnol nd 1 ml of 5.7% sodium hydroxide were dded. The result ws recorded fter 5 minutes. - Liebermnn-Burchrd rection for steroidl/triterpenoidl nucleus: 2 g of powdered smple ws extrcted with 500 ml of methnol. The extrct ws filtered nd the filterte ws gently concentrted to dryness on wter bth. 500 mg of the dried extrct ws dissolved in 2 ml of cetic nhydride nd llowed to cool. With the test tube inside ice pck nd slnted t n ngle of bout 45 degree, 2 ml of concentrted tetroxosulphte (VI) cid ws crefully poured by the side of the test tube. Colour obtined ws noted. Blue-green ring indicted the presence of terpenoids. -Keller-Kilini test for de-oxy sugrs in crdic glycosides: A methnol extrct ws obtined nd the extrct reduced to dryness. 50 mg of this ws dissolved in 2 ml chloroform. Tetroxosulphte (VI) cid ws dded to form lyer nd the colour t interphse recorded. - Legl test: The extrct ws dissolved in pyridine nd 5 drops of 2% sodium nitroprusside together with 4-5 drops of 20% sodium hydroxide were dded. Deep colour indicted the presence of crdenolides. - Slkowski s test: 200 mg of the extrct ws dissolved in 2 ml of chloroform. Concentrted tetroxosulphte (VI) cid ws crefully dded to form lower lyer. A reddish-brown t the interfce indicted the presence of steroidl ring (i.e. glycone portion of the crdic glycoside) 11

4 Hypoglycemic effects of the queous seed extrct of Hunteri umbellt Reducing sugrs: - Fehling s test: Wter extrct of the powdered mteril ws obtined by boiling on wter bth. To 2 ml of the extrct, in the test tube ws dded, 1 ml ech of Fehling s solutions A nd B. The mixture ws shken nd heted in wter bth for 10 minutes. The colour obtined ws recorded. Sponins: - Frothing test: Wter extrct ws obtined by boiling on wter both. The extrct ws trnsferred into test tube nd shken vigorously then ws left to stnd 10 minutes nd the result noted. Frothing persistence ment sponins were present. Flvonoids: 200 mg of the powdered smple ws boiled in 10 ml of bsolute ethnol for 10 minutes. The solution ws llowed to cool nd then filtered. To 2 ml of the filterte ws dded concentrted hydrochloric cid nd mgnesium ribbon. Pink-tomto red colour indicted the presence of flvonoids. Anthrquinone derivtives: Bontrger s test: Chloroform extrct of the powdered smple ws obtined by boiling on the wter bth. To 2 ml of this extrct, 1 ml of dilute (10%) mmoni ws dded nd the mixture ws shken. Any colour chnge ws recorded. Acute extrct tretment in orl glucose-induced hyperglycemic rts In the high orl glucose hyperglycemic model, hr fsted rts were rndomly llotted to 6 groups of 6 rts per group such tht the difference within nd between groups does not exceed ±20% of the verge weight of smple popultion of rts. Group I rts, which served s the untreted control, were orlly pretreted with 10 ml/kg of distilled wter 1 hr before tretment with nother 10 ml/kg/orl of distilled wter. Group II rts served s the model control nd were pretreted with 10 ml/kg/orl of distilled wter 1 hr before orl tretment with 3 g/kg of D-glucose (Anlr). Groups III - VI rts which served s the tretment groups were orlly pretreted with 1 mg/kg of glibenclmide (Donil, Hoechst Mrion Roussel Limited, Mumbi, Indi), 50 mg/kg, 100 mg/kg, nd 200 mg/kg of HU, respectively, for 1 hr before the orl dministrtion of 3 g/kg of D-glucose, s described by Sepici et l. (2004). Acute extrct tretment in nicotine-induced hyperglycemic rts Sme experimentl protocol described for high orl glucose-induced hyperglycemic model ws dopted for nicotine-induced hyperglycemi except tht orl glucose tretment ws replced with 50 µg/kg of nicotine dministered s described by Ald (2001) nd tht nicotine ws injected vi the intrperitonel route. Repeted extrct tretment in normoglycemic rts In the repeted dose model, hr fsted rts were rndomly llotted to 5 groups of 6 rts per group such tht the difference within nd between groups does not lso exceed ±20% of the verge weight of smple popultion of rts. Group I rts, which served s the untreted control, were orlly treted with 10 ml/kg of distilled wter while Groups II - V tht served s the tretment groups, were treted with single, dily orl 50 mg/kg, 100 mg/kg, 200 mg/kg of HU for 14 dys, respectively. Mesurement of fsting blood glucose in cute nd repeted extrct treted rts Blood smple from the rt til vein for fsting whole blood glucose ws collected by til tipping method. The til ws gently squeezed to let out 2-3 drops of fresh whole blood which were plced on the test spot of the glucose strip fter which the test strip is gently inserted into Test Strip Pltform of the Microprocessor digitl blood glucometer nd the redings were recorded (World Helth Orgniztion, 1980). The blood glucose monitor ws clibrted nd vlidted t the beginning of, midwy into nd t the end of the experiment, using stndrd lbortory method of spectrophotometry for the determintion of blood glucose concentrtions s previously described by Ajl et l. (2003). Blood glucose concentrtion ws estimted using glucose oxidse method of Trinder (1969) on One Touch Bsic Blood Glucose Monitoring System (LifeScn Inc., Milpits, Cliforni, U.S.A.). For the cute extrct treted hyperglycemic models, blood glucose concentrtions were determined t 0 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, nd 6 hr, respectively. For the repeted dose model, the fsting blood glucose concentrtions were determined on the 1 st nd 15 th dy of the experiment following n overnight fst. Dt Anlysis Results were presented s men ± S.D. for body weights while dt for biochemicl indices were expressed s men ± S.E.M. of six observtions. Sttisticl nlysis ws done using two-wy nlysis of vrince followed by post-hoc test, Student-Newmn-Keuls test on SYSTAT Sttisticl significnce were considered t p<0.05, p<0.01, nd p< Results nd Discussion Severl in vivo cute nd chronic drug-induced hyperglycemic models hve been developed nd used to investigte the hypoglycemic effects of medicinl plnts with ntidibetic potentils. The models include orl glucose loding- nd nicotine-induced hyperglycemi (Gryson nd Oyebol, 1985). Other gents tht hve been used in vrious hyperglycemic/dibetic models include dizoxide, lloxn, streptozotocin (Adeneye nd 12

5 Adeneye nd Adeyemi Agbje, 2008); drenline (Oyebol nd Durosiye, 1988); nd glucgon, growth hormone, thyroxine, nd cortisol (Ald et l., 2005). Glucose is the simplest metbolic end-product of crbohydrte metbolism which is most redily bsorbed into the port blood from the gstro-intestinl trct following its orl ingestion (Guyton, 1991). Its postbsorptive stte is mrked by postpndril hyperglycemic stte which often is ccompnied by incresed pncretic insulin secretion (hyperinsulinemi), prticulrly in the first few hours postpndril (Guyton, 1991b). In the present study, single high orl glucose tretment ws ssocited with significnt hyperglycemi in the distilled wter-pretreted, high glucose-treted rts, prticulrly within the first 2 hr post-orl dministrtion (Figure 2). However, the blood glucose levels stedily decresed over the succeeding 5 hr reching significnt (p<0.05) hypoglycemic levels t 5 th - 6 th hr. (Figure 2). Blood glucose conc. (mg/dl) f d d d b b b c c 0 hr 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr Time course (hour) Group I Group II Group III Group IV Group V Group VI, b nd c represent significnt decreses t p<0.05, p<0.01 nd p<0.001, respectively, when compred to the bsl vlues t 0 hr while d, e nd f represent significnt increses t p<0.05, p<0.01 nd p<0.001, respectively, when compred to bsl control vlues t 0 hr Group I = 10 ml/kg of orl distilled wter Group II = 10 ml/kg of orl distilled wter + 3 g/kg/orl of D-glucose Group III = 1 mg/kg of glibenclmide + 3 g/kg/orl of orl D-glucose Group IV = 50 mg/kg/dy of Hunteri umbellt queous seed extrct + 3 g/kg/orl of orl D-glucose Group V = 100 mg/kg/dy of Hunteri umbellt queous seed extrct + 3 g/kg/orl of orl D-glucose Group VI = 200 mg/kg/dy of Hunteri umbellt queous seed extrct + 3 g/kg/orl of orl D-glucose Figure 2. Effects of single orl tretment with 10 ml/kg of distilled wter, 1 mg/kg of glibenclmide nd mg/kg of Hunteri umbellt queous seed extrct on the fsting blood glucose (mg/dl) in glucose-induced hyperglycemic rts Orl pretretment with glibenclmide, on the other hnd, ws not ssocited with ny significnt (p>0.05) ltertions in the blood glucose concentrtion when compred to bsl vlue t 0 hr. Over the succeeding 5 hr, the blood glucose concentrtions in the glibenclmide nd mg/kg of HU pretreted rts, significntly (p<0.05, p<0.01, nd p<0.001) nd progressively decresed, becoming most significntly (p<0.00l) reduced t the 5 th nd 6 th hr when compred to the vlues t 1 hr. However, the hypoglycemic effect of HU t 200 mg/kg t 2-6 hr ws comprble to tht of glibenclmide (Figure 2). As shown in figure 2, the significnt (p<0.05, p<0.01, nd p<0.001) dose relted ttenution of significnt rise in the post-bsorptive blood glucose concentrtions in the glibenclmide- nd HU-pretreted rts is suggestive tht glibenclmide nd HU could be mediting their hypoglycemic ction vi inhibition of intestinl glucose uptke, mechnism which is similr to those of α-glucosidse inhibitors (e.g. crbose). The significnt (p<0.05) 5 th -6 th hr postpndril hypoglycemi recorded in the distilled wter-pretreted, glucose loded rts could be due to the secondry effect of hyperinsulinemi following the glucose lod. This is well estblished physiologicl response to hyperglycemi following high glucose lod or intke (Hedeskov, 1980). Literture hs shown tht extrcts with high triterpenoid sponins content medite their hypoglycemic effect vi inhibition of intestinl glucose uptke, incresed heptic glucose deposition nd enhnced hyperinsulinemi (Shne-McWhorter, 2001). 13

6 Hypoglycemic effects of the queous seed extrct of Hunteri umbellt Therefore, it is possible for the sponins contined in HU be responsible for the observed ttenution in the postbsorptive glucose concentrtion. However, further studies will be required to vlidte this proposed hypothesis. Nicotine, n lkloid nd the ctive principle contined in tobcco is usully implicted in cusing the cute biologicl effects of tobcco smoking (Assli et l., 1999). It hs been reported to induce hyperglycemi in cnines (Milton, 1966; Gryson nd Oyebol, 1985) nd rodents (Oyebol nd Ald, 1993; Ald, 2001). This effect is believed to be due to its modultory ction on drenergic mechnisms by stimulting the nicotinic cetylcholine receptors (Westfll, 1965). Thus, nicotine-induced hyperglycemi s model of cute druginduced hyperglycemi is well estblished (Oyebol nd Ald, 1993). 160 Blood glucose conc. (mg/dl) f d d d b bb b b c c Group I Group II Group III Group IV Group V Group VI 0 0 hr 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr Time course (hours), b nd c represent significnt decreses t p<0.05, p<0.01 nd p<0.001, respectively, when compred to the bsl vlues t 0 hr while d, e nd f represent significnt increses t p<0.05, p<0.01 nd p<0.001, respectively, when compred to bsl vlues t 0 hr Group I = 10 ml/kg of orl nd intrperitonelly injected distilled wter, respectively Group II = 10 ml/kg of orl distilled wter + 50 µg/kg/intrperitonel route of nicotine Group III = 1 mg/kg of glibenclmide + 50 µg/kg/intrperitonel route of nicotine Group IV = 50 mg/kg/dy of Hunteri umbellt queous seed extrct + 50 µg/kg/intrperitonel route of nicotine Group V = 100 mg/kg/dy of Hunteri umbellt queous seed extrct + 50 µg/kg/intrperitonel route of nicotine Group VI = 200 mg/kg/dy of Hunteri umbellt queous seed extrct + 50 µg/kg/intrperitonel route of nicotine Figure 3. Effects of single orl tretment with 10 ml/kg of distilled wter, 1 mg/kg of glibenclmide nd mg/kg of Hunteri umbellt queous seed extrct on the fsting blood glucose (mg/dl) in nicotine-induced hyperglycemic rts In the present study, significnt (p<0.001) hyperglycemi ws relibly estblished within 1 hr following the intrperitonel injection of 50 µg/kg of nicotine, which lsted over the next hour (Figure 3). However, the hyperglycemi ws significntly ttenuted in rts pretreted with glibenclmide nd vrying doses of HU in dose relted fshion, mking the blood glucose fll within the rnge of bsl vlue t the 2 nd hr but significntly (p<0.05, p<0.01 nd p<0.001) lower thn the bsl vlue t 0 hr t 3 rd to the 6 th hr of the study (Figure 3). This is n indiction tht the hypoglycemic effect of HU becme more pronounced within the 3 rd to the 6 th hr postdministrtion, n effect which ws lower thn tht of the reference drug, glibenclmide. Thus, from the forementioned, it is cler tht HU prt from inhibiting intestinl glucose uptke, it could lso be mediting its hypoglycemic effect vi inhibition of the drenergic homeosttic mechnism on cute course. In the repeted dose study, mg/kg/dy of HU produced sustined nd significnt (p<0.05, p<0.01, nd p<0.001) hypoglycemic effect when compred to the untreted control vlues (Figure 1). 14

7 Adeneye nd Adeyemi Fsting blood glucose (mg/dl) Group I Group II Group III Tretment groups b c Group IV FBG on dy 1 FBG on dy 15, b nd c represent significnt reductions t p<0.05, p<0.01 nd p<0.001 when compred to untreted control vlues (Group I). Group I = 10 ml/kg/dy of orl distilled wter Group II = 50 mg/kg/dy of Hunteri umbellt queous seed extrct Group III = 100 mg/kg/dy of Hunteri umbellt queous seed extrct Group IV = 200 mg/kg/dy of Hunteri umbellt queous seed extrct Figure 1. Effect of repeted orl tretment with mg/kg/dy of Hunteri umbellt queous seed extrct on the fsting blood glucose (mg/dl) in normoglycemic rts fter 14 dys of tretment The observed hypoglycemic effects of HU extrct could be due to the presence of ny or combintion of the ctive principle(s) contined in the extrct. Tble 1: Phytochemicl constituents of the queous seed extrct of Hunteri umbellt (HU) Tests Results Tnnins (i) Ferric chloride test (for ctechol tnnin) +++ (ii) Bromine wter test (for condensing tnnin) ++ Phlobtinnins + Alkloids (i) Drggendoff s test +++ (ii) Myer s regent ++ (iii) Wgner s test +++ Crdic glycosides (i) Keller-Kilini test +++ (ii) Kedde test ++ (iii) Legl s test ++ (iv) Slkowski test ++ (v) Liebermnn-Burchrd s test +++ Reducing sugr (i) Fehling A nd B test (for Hexose sugr) + (ii) Resorcinol test (for Ketosugr) + (iii) Phloroqlucinol test (for Pentosugr) ++ (iv) Brfoed test (for Monoscchrides) + Flvonoids (i) Led cette test +++ (ii) Ferric chloride test (for phenolic nucleus) +++ (iii) Sodium chloride test +++ Sponins (i) Benedict s test ++ (ii) Emulsion test ++ (iii) Frothing test ++ Anthrquinone (i) Borntrger s test (for free nthrquinones) +++ Anthocynosides test - -: not detected; +: present in low concentrtion; ++: present in moderte concentrtion; +++: present in high concentrtion. 15

8 Hypoglycemic effects of the queous seed extrct of Hunteri umbellt Results of the phytochemicl studies showed HU to contin high concentrtion of tnnins, flvonoids, lkloids, nthrquinone nd crdic glycosides, while phlobtinnins nd sponins were present in low nd moderte concentrtions, respectively. However, nthocynosides were bsent in the plnt extrct (Tble 1). It is well documented in the literture tht medicinl plnts with hypoglycemic nd ntidibetic effects usully contin high concentrtions of lkloids, flvonoids (Oldele et l., 1994; Ro nd Ro, 2001; Shrm et l., 2008), steroid glycosides (Ivorr et l., 1989; Adllu nd Rdhik, 2000) nd terpenoids (Reher et l., 1991, Shne-McWhorter, 2001). The presence of these phytocompounds in high concentrtions could ccount for the significnt hypoglycemic effect of the extrct, either singly or in synergy with one nother. Agin, this hypothesis will require further vlidtion. Aprt from the forementioned hypoglycemic mechnisms, it is lso possible tht HU could be mediting its effects vi hyperinsulinemi [which itself, could be cused by incresing secretion of insulin from the pncretic cells of islets of Lngerhns or its relese from bound insulin (Pri nd Amrnth, 2004)], incresed peripherl utiliztion of glucose or combintion of ny of the stted mechnisms. In the current study, these possible mechnisms were not investigted but could constitute res of further studies. Tble 2: Sequence nd result of the min test of Up-nd-Down Procedure of cute orl toxicity of Hunteri umbellt queous seed extrct in treted rts Test Animl Dose Short-term Long-term Sequence Identity (mg/kg/orl) Outcome Outcome (the first 48 hr) (successive 12 dys) 1 B 175 survivl survivl 2 F 550 survivl survivl 3 I 2000 deth 4 A 550 survivl survivl 5 G 2000 deth 6 O 550 survivl survivl 7 L 2000 deth Tble 2 shows the sequence nd results of the min test of the Up nd Down Procedure of the cute orl toxicity. As shown in Tble 2, cute orl tretment t 2000 mg/kg cused deth in the rts for less thn 1 hr posttretment. Deth, on ech occsion, ws preceded by restlessness, periorl tremor, polypnoe nd generlized convulsion. On stepping down the dose to 550 mg/kg, there ws no ssocited deth but there were short-term (48 hr) behviourl toxicities which included hyperctivity succeeded by generlized musculr rigidity, piloerection, nd bilterl nrrowing of the eyelids. The delyed behviourl toxicities include decresed feeding pttern, progressive weight loss nd lopeci. Bsed on the computer-generted result, the clculted LD 50 vlue ws 1020 mg/kg/orl route. According to the Americn Society for Testing nd Mterils (1987), ny chemicl substnce with LD 50 estimte less thn 2000 mg/kg/orl route but greter thn 1000 mg/kg/orl could be considered to be slightly toxic, lthough Clrke nd Clrke (1977) consider ny compound with n estimted LD 50 greter thn 1000 mg/kg/orl to be sfe. Thus, the result of the cute orl toxicity of HU suggests it could be toxic t high dose on cute exposure. The effect of HU on the body weight is lso significnt. Tble 3 shows effect of repeted, single dily orl dministrtion of HU on the initil, finl nd the percentge weight chnge of verge body weight of treted rts. As shown in the tble, orl dministrtion of mg/kg of HU for 14 dys ws ssocited with significnt (p<0.05 nd p<0.01) dose relted weight loss when compred to tht of untreted control (Group I) rts. 16

9 Adeneye nd Adeyemi Tble 3: Effect of 14 dys of repeted orl tretment with 10 ml/kg/dy of distilled wter, mg/kg/dy of Hunteri umbellt queous seed extrct on the verge body weight in norml rts Tretment Initil weight (g) Finl weight (g) Percentge weight chnge Groups (on dy 1) (on dy 15) (%) I ± ± ± 10.3 II ± ± ± 7.6 III ± ± ± 7.3 b IV ± ± ± 4.3 b nd b represent significnt decreses t p<0.05 nd p<0.01, respectively, when compred to untreted control vlues (group I) Group I = 10 ml/kg/dy of orl distilled wter Group II = 50 mg/kg/dy of Hunteri umbellt queous seed extrct Group III = 100 mg/kg/dy of Hunteri umbellt queous seed extrct Group IV = 200 mg/kg/dy of Hunteri umbellt queous seed extrct The observed significnt weight loss nd ssocited hypoglycemi could not hve been medited vi ppetite inhibiting mechnism becuse there ws no chnge in the feeding pttern of HU-treted rts. However, the exct mechnism by which the extrct induced progressive weight is currently being investigted in our lbortory. In summry, the present study hs shown tht the queous seed extrct of Hunteri umbellt cused significnt reductions in norml rts nd the two experimentl models of cute drug-induced hyperglycemi in rts, which were medited vi glucose uptke inhibition nd drenergic inhibition. Further studies on other hypoglycemic mechnisms prt from the forementioned nd identifiction of suspected hypoglycemic phytocomponents in HU re currently on-going in our reserch lbortory. Also, results obtined in this preliminry study will form templte for subsequent studies on the hypoglycemic nd weight loss effects of HU s well s the mechnisms involved. Acknowledgements The uthors wish to cknowledge the technicl support of Mr. T.I. Adeleke, Senior Technologist, in the Deprtment of Phrmcognosy, Fculty of Phrmcy, College of Medicine, University of Lgos, Idi-Arb, Lgos Stte, Nigeri. References Acute Orl Toxicity (OECD Test Guideline 425) (AOT) Sttisticl Progrmme (AOT425SttPgm), Version ,FF.html. Adllu B, Rdhik B Hypoglycemic, diuretic nd hypocholesterolemic effect of winter cherry (Winthni somnifer, Dunl) root. Indin Journl of Experimentl Biology 38: Adeneye AA, Amole OO, Adeneye AK Hypoglycemic nd hypocholesterolemic ctivities of the queous lef nd seed extrct of Phyllnthus mrus in mice. Fitoterpi 77: Adeneye AA, Ajgbonn OP, Adeleke TI, Bello SO. 2006b. Preliminry toxicity nd phytochemicl studies of the stem brk queous extrct of Musng cecropioides in rts. Journl of Ethnophrmcology 105: Adeneye AA, Agbje EO Phrmcologicl evlution of orl hypoglycemic nd ntidibetic effects of fresh leves ethnol extrct of Morind lucid Benth. in norml nd lloxn-induced dibetic rts. Africn Journl of Biomedicl Reserch 11: Ajl MO, Fsnmde A, Mgbgbeol O, Smuel O, Afonj O Serum free lph-tocopherol (nti-oxidnt) in non-insulin dependent dibetes mellitus. Africn Journl of Endocrinology nd Metbolism 2: Ald RA Effects of clcium chnnel blockers on nicotine-induced hyperglycemi in the rt. Africn Journl of Medicine nd Medicl Sciences 30: Ald, ARA, Flokun PO, Oyebol DDO Intestinl glucose uptke in norml, untreted nd insulin-treted dibetic dogs. Africn Journl Medicine nd Medicl Sciences 34: Americn Society for Testing nd Mterils Stndrd Test Method for EstimtingAcute Orl Toxicity in Rts. Americn Society for Testing nd Mterils E , Phildelphi, U.S.A. Amos AF, McCrthy DJ, Zimmet P The rising globl burden of dibetes nd its complictions: estimtes nd projections to the yer Dibetic Medicine 14: S7 - S85 Assli AR, Beigel Y, Schreibmn R, Schfer Z, Finru M Weight gin nd insulin resistnce during nicotine replcement therpy. Clinicl Crdiology 22: Clrke EGC, Clrke ML Veterinry Toxicology. London: Csell nd Collier Mcmilln Publishers p. 17

10 Hypoglycemic effects of the queous seed extrct of Hunteri umbellt Flodun A, Nworgu Z, Ikponmwons MO Phytochemicl components of Hunteri umbellt (K. Schum) nd its effect on isolted non-pregnnt rt uterus in oestrus. Pkistni Journl of Phrmceuticl Sciences 19(3): Gill LS Ethnomedicl uses of plnts in Nigeri. Benin-City: Uniben Press. 134 p. Gryson J, Oyebol DDO Effect of nicotine on blood flow, oxygen consumption nd glucose uptke in the cnine smll intestine. British Journl of Phrmcology 85: Guyton AC Digestion nd Absorption in the Gstrointestinl Trct. In: Textbook of Medicl Physiology. 8 th edition. Phildelphi: W.B. Sunders p. Guyton AC. 1991b. Secretory Functions of the Alimentry Trct. In: Textbook of Medicl Physiology. 8 th edition. Phildelphi: W.B. Sunders p. Hedeskov CJ Mechnism of glucose-induced insulin secretion. Physiologicl Revolutions 60: Hutchinson J, Dlziel JM Hunteri. In: Flor of West Tropicl Afric. Vol. 2, 2 nd edition. Kew: Royl Botnic Grdens p. Ivorr MD, Py M, Villr A A review of nturl products nd plnts s potentil ntidibetic drugs. Journl of Ethnophrmcology 27: Kld R, Rätsep A, Lember M Predictors of qulity of life of ptients with type 2 dibetes. Ptient Preferences nd Adherence 2: King H, Albert RE, Hermn WH Globl Burden of Dibetes : Prevlence, Numericl estimtes nd Projections. Dibetes Cre 21(9): Milton AS The effect of nicotine on blood glucose level nd plsm non-esterified ftty cids levels in intct nd drenlectomized cts. British Journl of Phrmcology 26: Murry M, Pizzorno J Encyclopedi of Nturl Medicine, 2 nd edition. Rockling: Prim Helth. 401 p. Oldele SB, Ayo JO, Adudi AO Medicinl nd physiologicl properties of flvonoids, coumrin derivtives nd nthrquinones of plnt origin. West Africn Journl of Phrmcology nd Drug Reserch 11: Oput RN Mlnutrition relted dibetes mellitus (MRDM). Nigerin Journlof Endocrinology nd Metbolism 3(1): Oyebol DDO, Ald RA Effects of drenergic receptor blockers on drenline nd nicotine-induced hyperglycemi in the rt. Africn Journl of Medicl Sciences 22: Pri L, Amrnth S Antidibetic ctivity of Boerhvi diffus L. Effect on heptic key enzymes in experimentl dibetes. Journl of Ethnophrmcology 91: Ro BK, Ro CH Hypoglycemic nd ntihyperglycemic ctivity of Syzygium lternifolium (Wt.) Wlp. seed extrcts in norml nd dibetic rts. Phytomedicine 8: Reher G, Slijepcevic M, Krus L Hypoglycemic ctivity of triterpenes nd tnnins from Srcopoterium spinosum nd two Snguisorb species. Plnt Medic 57: A57 - A58. Rotshteyn Y, Zito SW Appliction of modified in vitro screening procedure for identifying herbls possessing sulfonylures-like ctivity. Journl of Ethnophrmcology 93: Sepici A, Gürbüz I, Çevik C, Yesild E Hypoglycemic effects of myrtle oil in norml nd lloxn-dibetic rbbits. Journl of Ethnophrmcology 93: Shne-McWhorter L Biologicl complementry therpies: focus on botnicl products in dibetes. Dibetes Spectrum 14: Shrm B, Blomjumder C, Roy P Hypoglycemic nd hypolipidemic effects of flvonoid rich extrct from Eugeni jmboln seeds on streptozotocin induced dibetic rts. Food nd Chemicl Toxicology 46(7): Sofowor A Medicinl Plnts nd Trditionl Medicine in Afric. 2 nd edition. Ibdn: Spectrum Books Ltd. 150 p. Trese GE, Evns WC A Textbook of Phrmcognosy, 13 th edition. London: Billiere Tindll Ltd. Trinder P Determintion of Blood Glucose using 4 minophenzone s Oxygen Acceptor. Journl of Clinicl Pthology 22: United Sttes Ntionl Institutes for Helth publiction no Westfll TC Tobcco lkloids nd the relese of ctecholmines. In: Tobcco nd relted compounds,1 st edition. USA: Von Euler Pergmon Press p. World Helth Orgniztion The WHO expert committee on dibetes mellitus. Technicl Report Series No World Helth Orgniztion Dibetes mellitus. Fct sheet number 138 nd 236, Genev. World Helth Orgniztion WHO lunches the first globl strtegy on trditionl medicine: Press relese WHO/ 18