Journal of the Science of Food and Agriculture J Sci Food Agric 88: (2008)

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1 Journl of the Science of Food nd Agriculture J Sci Food Agric 88: (28) Protective effect of polyphenol-rich extrct prepred from Mlysin coco (Theorom cco) on glucose levels nd lipid profiles in streptozotocin-induced dietic rts Azli Mohd Mokhtr Ruzidi, 1,2 Mleyki Mhd Jlil Ae, 1 Ismil Amin, 1 Adul Ghni Nwlyh 1 nd Hmid Muhjir 3 1 Deprtment of Nutrition nd Dietetics, Fculty of Medicine nd Helth Sciences, Universiti Putr Mlysi, 434 Serdng, Selngor, Mlysi 2 Biotechnology Reserch Institute, Universiti Mlysi Sh, Kot Kinlu, Sh, Mlysi 3 Deprtment of Microiology, Fculty of Biotechnology nd Biomoleculr Sciences, Universiti Putr Mlysi, 434 Serdng, Selngor, Mlysi Astrct BACKGROUND: Coco ens re used for prepring coco liquor nd coco powder, which re the min ingredients of coco-sed products. Previous studies hve reported the helth enefits of coco polyphenols in reducing the risk of crdiovsculr diseses. However, there is no report on the efficcy of coco polyphenols on dietes mellitus. Therefore this study ws designed to evlute the protective effect of coco polyphenol-rich extrct (CE) on glucose levels nd lipid profiles in streptozotocin (STZ)-induced dietic rts. Mle Sprgue- Dwley rts were divided into dietic control, dietic CE nd dietic glienclmide groups. RESULTS: Three different dosges of CE (1, 2 nd 3 mg per 1 g ody weight) were dministered orlly once dy for 1week efore STZ injection nd for 3weeks therefter. The results showed tht CE could normlise the ody weight loss cused y STZ. In the 2 mg CE-pretreted group there ws 143% increse in plsm glucose levels, compred with 226% increse in dietic control rts. CE could lso normlise totl cholesterol, triglycerides nd high-density lipoprotein cholesterol t the end of the experiment compred with the seline. CONCLUSION: The present study suggests tht pretretment with CE from rosted coco ens could prevent the development of dietes induced y STZ injection in rts. 28 Society of Chemicl Industry Keywords: Theorom cco; coco extrct; glucose levels; hypoglycemic INTRODUCTION There re severl wys of preventing dietes nd/or controlling its progression. Pulic nd professionl wreness of the risk fctors nd symptoms of dietes is n importnt step towrds its prevention nd control. There is incresing demnd y ptients for nturl products with ntihyperglycemic ctivity owing to the side effects ssocited with insulin nd orl hypoglycemic drugs. 1,2 Therefore it hs ecome necessry to look for n economicl s well s therpeuticlly effective use of nturl products in prevention nd tretment, especilly in developing nd underdeveloped countries. The serch for sfer nd more effective compounds to protect β-cells from inflmmtory destruction is still in progress. Severl compounds such s metllothionein, nicotinmide nd ( )-epictechin hve een reported to inhiit the dietogenic ction of streptozotocin (STZ) or lloxn in niml studies. 3,4 Plm Vitee (plm oil vitmin E) hs protective ction ginst the toxic inflmmtion cused y STZ. 5 Coco ens hve een reported to e rich source of polyphenols, especilly ( )-epictechin. Some of the erliest studies estlished tht the mjor flvnoids in coco ens were ctechin, epictechin, the dimers epictechin-(4β 8)-ctechin (procynidin B-l) nd epictechin-(4β 8)-epictechin (procynidin B-2) nd the trimer [epictechin-(4β 8)] 2 - epictechin (procynidin C-l). 6,7 In previous study we showed tht diet contining coco polyphenolrich extrct reduced the glucose levels nd lipid profiles in STZ-induced dietic rts. 8 In similr work, Correspondence to: Ismil Amin, Deprtment of Nutrition nd Dietetics, Fculty of Medicine nd Helth Sciences, Universiti Putr Mlysi, 434 Serdng, Selngor, Mlysi E-mil: min@medic.upm.edu.my (Received 3 July 27; revised version received 21 Jnury 28; ccepted 21 Jnury 28) Pulished online 17 April 28; DOI: 1.12/jsf Society of Chemicl Industry. J Sci Food Agric /28/$3.

2 Protective effect of polyphenol-rich coco extrct in dietic rts Oske et l. 9 demonstrted tht pronthocynidins derived from coco inhiited dietes-induced ctrct formtion. However, limited reserch hs een conducted on the protective effect of coco polyphenol-rich extrct (CE) ginst STZ dietogenic ction. Therefore the present study ws focused on evluting the protective ction of CE ginst the destruction of insulin-producing β-cells of the pncres in STZ-induced dietic rts. MATERIALS AND METHODS Preprtion of ethnolic extrct Rw (fermented nd dried) Mlysin coco ens were purchsed from KL-Kepong Coco Products Sdn. Bhd. (Port Klng, Selngor, Mlysi). The ens were rosted in n ir oven for 2 min t 14 C. 1 After cooling to room temperture, the rosted ens were deshelled using coco reker (Limprimit, John Gordon & Co., Lneshire UK). The cotyledons were ground nd deftted with petroleum ether (.p. 4 6 C) in Soxhlet pprtus. The deftted cotyledons were ir dried to remove the solvent residue. The extrct ws prepred y treting the deftted powder with 8% (v/v) ethnol for 2 h. The ethnol residue ws removed from the extrct using rotry evportor (Rotvor R-2, Büchi, Flwil, Switzerlnd) for 2 min t 7 Cnd the resulting extrct ws lyophilised. This ethnolic extrct ws considered to e coco polyphenol-rich 8 nd ws used for totl phenolic determintion nd the niml study. Determintion of totl phenolics Totl phenolic content ws estimted ccording to the Folin Cioclteu ssy. 11 Briefly, CE ws dissolved in 8% (v/v) ethnol nd centrifuged (Rotofix 32, Hettich Zentrifugen, Tuttlingen, Germny) t 1 g for 15 min. Following centrifugtion, 1 µl of the superntnt ws mixed with.75 ml of Folin Cioclteu regent (previously diluted 1:1 with distilled wter) nd llowed to stnd t room temperture for 5 min. Sodium cronte solution (.75 ml) ws then dded to the mixture. After stnding for further 9 min t room temperture, the sornce t 725 nm ws recorded using UV visile spectrophotometer (Anthelie Advnced 5, Secomm, Ales, Frnce). A stndrd clirtion curve ws constructed using.2.12 mg ml 1 ( )- epictechin (Sigm, St Louis, MO, USA). Results were expressed s mg epictechin equivlents g 1 extrct. Animl study Preprtion of nimls This study hs een pproved y the Animl Cre nd Use Committee of the Fculty of Medicine nd Helth Sciences, Universiti Putr Mlysi. Fifty mle Sprgue-Dwley rts (2 35 g initil weight) were purchsed from Syrikt Ush Chy Sdn. Bhd. (Btu Cves, Selngor, Mlysi). The rts were housed in individul plstic cges with stinless steel covers nd kept t room temperture (24 28 C) under 12/12 h drk/light cycle. Animls were llowed free ccess to their respective diets nd wter. All rts were llowed 7 dys to dpt to the environment efore eing given the tretment. The experiment ws conducted for 28 dys. Body weights, food intkes nd lood glucose levels were recorded weekly. The rts were divided into five groups, ech consisting of ten rts (n = 1): group 1 dietic rts dministered norml sline (DC); group 2 dietic rts dministered 1 mg ml 1 CE (DCE1); group 3 dietic rts dministered 2 mg ml 1 CE (DCE2); group 4 dietic rts dministered 3 mg ml 1 CE (DCE3); group 5 dietic rts dministered 1 mg ml 1 glienclmide (DG). CE (1, 2 nd 3 mg ml 1 ) nd glienclmide (1 mg ml 1 ) were suspended in.9% (w/v) norml sline nd given dily (1 ml per 1 g ody weight) to the experimentl rts y gstric intution using force-feeding needle. The nimls were given CE once dily for 7 dys efore STZ injection nd for 21 dys therefter. At dy 7 the rts were given CE 1 h efore STZ injection. Induction of dietes STZ (Sigm) ws used for inducing dietes in the rts t dy 7. After overnight fsting, the rts were injected intrvenously with 45 mg kg 1 ody weight of STZ dissolved in.5 mol L 1 citrte uffer (ph 4.5). Rts injected with the sme volume of.5 mol L 1 citrte uffer served s the control group. Determintion of glucose levels nd lipid profiles At dys, 9 nd 28 of the experiment, 5 ml of lood ws collected from the dominl ort of ech niml, plced in Vcutiner tue nd centrifuged (Universl 32, Hettich Zentrifugen) t 1 g for 1 min t room temperture. The superntnt ws collected nd kept t 2 C for further nlysis. Plsm glucose levels nd lipid profiles were mesured using chemistry nlyser (Automtic Anlyser 92, Hitchi, Tokyo, Jpn). Sttisticl nlysis Dt were expressed s men ± stndrd error of men (SEM). One-wy nlysis of vrince (ANOVA) ws pplied to determine differences etween groups. Duncn s multiple rnge test ws used to find significnt differences mong mens. Results were considered significntly different t the level of P <.5. J Sci Food Agric 88: (28) 1443 DOI: 1.12/jsf

3 AMM Ruzidi et l. RESULTS The initil ody weights of rts were in the rnge 2 35 g. The ody weights of ech group of rts were not significntly different efore STZ injection (Tle 1). At 21 dys fter STZ injection the ody weights of DC, DCE1, DCE3 nd DG rts were significntly decresed (P <.5) compred with their initil weights. However, there ws no significnt decrese in ody weight in the DCE2 group. All rts exhiited decrese in ody weight gin fter STZ injection t week 1 (Fig. 1). The ody weights of DC nd DG rts were drsticlly decresed t week 2. However, in the CE-pretreted groups (DCE1, DCE2 nd DCE3) the ody weight loss ws much lower compred with the DC group t weeks 2, 3 nd 4, though there ws no significnt difference. Figure 2 shows the protective effect of CE on plsm glucose levels in STZ-induced dietic rts. At 2 dys fter STZ injection, i.e. t dy 9, glucose levels incresed significntly (P <.5) in ll groups compred with the initil glucose levels. In the CEpretreted groups (DCE1, DCE2 nd DCE3) nd the glienclmide-pretreted group (DG) the increse ws significntly lower (P <.5) compred with the DC group t dy 9. The increments in glucose levels in the DC, DCE1, DCE2, DCE3 nd DG groups were 226, 163, 143, 156 nd 148% respectively. There ws no significnt increse in glucose levels in treted rts t the end of the study (dy 28) compred with dy 9, except for the DCE3 group. Only the DCE2 group showed significnt decrese (P <.5) in glucose Tle 1. Effect of coco extrct (CE) on ody weight of rts Body weight (g) Group Initil Finl Dietic control (DC) ± ± 38. Dietic + 1 mg CE (DCE1) ± ± 26.3 Dietic + 2 mg CE (DCE2) ± ± 29.2 Dietic + 3 mg CE (DCE3) 32.8 ± ± 8.2 Dietic + glienclmide (DG) ± ± 26.9 Body weights were mesured weekly. Vlues re expressed s men ± SEM. Different letters indicte significnt differences (P <.5). Body weight gin (g) DC DCE1 DCE2 DCE3 DG Weeks Figure 1. Pttern of ody weight in control rts nd those pretreted with coco extrct (CE) nd glienclmide: DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. The coefficient of vrition for ll dt ws less thn 24%. Body weights were mesured weekly. Plsm Glucose Level (mmol L 1 ) d d cd c c c week week 1 week 4 d cd c Figure 2. Plsm glucose levels of dietic rts pretreted with coco extrct (CE): DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. Vlues with different letters re significntly different (P <.5) etween groups nd weeks. levels compred with the DC group t the end of the experiment (dy 28). The plsm totl cholesterol levels in ll groups were significntly higher (P <.5) t dy 9 fter STZ injection compred with dy (Fig. 3). At the end of the experiment (dy 28) the totl cholesterol levels in ll treted nimls were normlised. Figure 4 shows the effect of CE on plsm highdensity lipoprotein (HDL) cholesterol levels in STZinduced dietic rts. After STZ injection, ll rts exhiited significnt decrese (P <.5) in HDL cholesterol levels t dy 9 compred with dy. The reduction in HDL cholesterol levels ws in the rnge 54 61%. Interestingly, HDL cholesterol levels were normlised in CE- nd glyenclmide-pretreted rts t the end of the study. However, no significnt chnge in plsm low-density lipoprotein (LDL) cholesterol levels ws oserved during the 4 week experimentl period (Fig. 5). There were significnt increses (P <.5) in plsm triglyceride levels in DC, DCE1 nd DG rts fter STZ injection (Fig. 6). However, in the DCE2 Plsm Totl Cholesterol Level (mmol L 1 ) week week 1 week 4 Figure 3. Plsm totl cholesterol levels of dietic rts pretreted with coco extrct (CE): DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. Vlues with different letters re significntly different (P <.5) etween groups nd weeks J Sci Food Agric 88: (28) DOI: 1.12/jsf

4 Protective effect of polyphenol-rich coco extrct in dietic rts Plsm HDL-cholesterol Level (mmol L 1 ) c c c c c week week 1 week 4 c c c c Figure 4. Plsm HDL cholesterol levels of dietic rts pretreted with coco extrct (CE): DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. Vlues with different letters re significntly different (P <.5) etween groups nd weeks. Plsm LDL-cholesterol Level (mmol L 1 ) week week 1 week 4 Figure 5. Plsm LDL cholesterol levels of dietic rts pretreted with coco extrct (CE): DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. Vlues with different letters re significntly different (P <.5) etween groups nd weeks. nd DCE3 groups the elevtion ws significntly lower (P <.5) compred with the DC group t dy 9. At the end of the experiment normlistion of plsm triglyceride levels ws oserved in CE- nd glienclmide-pretreted rts. DISCUSSION STZ is specific β-cell toxin nd cn e used to chemiclly induce hyperglycemi in rts nd mice. It is tken up y pncretic β-cells vi glucose trnsporter (GLUT2) nd cuses lkyltion of deoxyrionucleic cid (DNA). 12,13 DNA dmge induces ctivtion of poly denosine diphosphte (ADP)-riosyltion, process tht is more importnt for the dietogenicity of STZ thn DNA dmge itself. 14 Poly ADP-riosyltion leds to the depletion of cellulr nicotinmide denine dinucleotide (NAD + ) nd denosine triphosphte (ATP). 15 Enhnced ATP dephosphoryltion fter STZ tretment supplies sustrte for xnthine oxidse (XOD), resulting in the formtion of superoxide rdicls. Consequently, Plsm Triglyceride Level (mmol L 1 ) c de e de cd c c cd week week 1 week 4 Figure 6. Plsm triglyceride levels of dietic rts pretreted with coco extrct (CE): DC, dietic control; DCE1, dietic + 1 mg CE; DCE2, dietic + 2 mg CE; DCE3, dietic + 3 mg CE; DG, dietic + glienclmide. Vlues with different letters re significntly different (P <.5) etween groups nd weeks. hydrogen peroxide nd hydroxyl rdicls re lso generted. Furthermore, STZ liertes toxic mounts of nitric oxide (NO), which inhiits conitse ctivity nd prticiptes in DNA dmge. 15 As result of the ction of STZ, β-cells undergo destruction y necrosis. In this study the totl polyphenol content of CE ws in the rnge mg epictechin equivlents g 1 extrct. A previous study showed tht coco ens re rich in polyphenols such s ( )-epictechin, (+)- ctechin, quercetin nd procynidin. 16 To evlute the protective effect of CE ginst STZ-induced dietes in rts, CE (1, 2 nd 3 mg ml 1 ) ws force-fed dily to the rts for 1 week efore STZ injection. On the lst dy of pretretment (dy 7), CE ws given to the rts 1 h efore STZ injection. This procedure ws sed on the findings of B et l., 17 which indicted tht ( )-epictechin metolite occurred t its mximum level in plsm etween 3 nd 6 min fter rts were given coco everge. Our results showed tht CE dministrtion significntly lowered (P <.5) the hyperglycemic ction of STZ in the DCE2 group. Tretment with CE lso seemed to prevent ody weight loss nd improve ody weight to some extent. Thus pretretment with CE could e effective in preventing the development of hyperglycemi following STZ injection. Kmtchouing et l. 18 lso reported tht Ancrdicee (Ancrdium occidentle) extrct showed protective effect ginst the dietogenic ction of STZ. Moreover, Gupt et l. 19 showed tht neem seed extrct hd protective effect on the hert nd erythrocytes of dietic rts. It is suggested tht CE my hve rected with or scvenged STZ. Superoxide dismutse (SOD) is n enzyme known to e prt of the ntioxidnt defence system of cells nd scvenger of free rdicls. Vucic et l. 2 reported tht the ctivity of SOD is low in dietes mellitus. CE my hve cted y incresing the resistnce of β-cells through ctivting SOD nd scvenging free rdicls cused y STZ. This scenrio is supported y Su et l., 21 who found tht green de c J Sci Food Agric 88: (28) 1445 DOI: 1.12/jsf

5 AMM Ruzidi et l. te polyphenols improved SOD levels in dietic rts. The ctul mechnisms of this phrmcologicl effect hve yet to e determined. The selection of the dose of glienclmide employed in this study ws sed on previous reserch y Ngpp et l. 22 Glienclmide is one of the most widely used orlly ctive drugs (sulfonylures) for the tretment of type 2 dietes mellitus. The cute hypoglycemic ction of glienclmide involves stimultion of insulin nd inhiition of glucgon secretion. 23 However, glienclmide is only effective when there re still surviving β-cells in the pncres. In the present study, glienclmide tended to lower plsm glucose levels, which my e due to ctivtion of pncretic β-cells to secrete insulin fter single dministrtion of glienclmide. It could e suggested tht the dietic rts in this study still hve some surviving β-cells in the pncres, though not sufficient to significntly decrese the plsm glucose levels. Generlly, dietic models re lso used to investigte successful tretments for hypercholesterolemi. 24 Anormlities in lipids nd lipoproteins ply key roles in the development nd progression of therosclerotic vsculr diseses in type 1 dietes mellitus. 25 The most common lipid normlities in dietes mellitus re chnges in plsm cholesterol nd triglyceride levels, which certinly contriute to the development of crdiovsculr diseses. 26 Hypercholesterolemi nd hypertriglyceridemi hve een reported to occur in STZ-induced dietic rts in severl studies. 24,27,28 As to the protective role of CE ginst STZ ction, rts pretreted with CE nd glienclmide did not seem to e protected from elevtion of plsm cholesterol levels y STZ. However, fter 3 weeks of further tretment with CE, totl cholesterol levels decresed significntly (P <.5) nd were normlised in dietic rts. In contrst, elevtion of triglyceride levels seemed to e significntly suppressed (P <.5) y pretretment with 2 nd 3 mg ml 1 CE in dietic rts. Thus the present study indictes tht CE exhiits protective effects on triglyceride levels in STZ-induced dietic rts. This study suggests tht polyphenols, the min components of CE, my e involved in the improvement of lipid profiles in dietic rts. The min cuse of elevted cholesterol nd triglyceride levels in STZinduced dietic rts is insulin deficiency. It is well known tht, under norml circumstnces, insulin ctivtes the enzyme lipoprotein lipse (LpL), which then hydrolyses very-low-density lipoprotein (VLDL) cholesterol. 29 However, in insulin-deficient dietic rts, LpL is not ctivted, resulting in hypercholesterolemi nd hypertriglyceridemi. Our present study lredy suggests tht the glucose-lowering effect of CE is due to the stimultion of insulin secretion in β-cells. This result is in greement with previous study which demonstrted tht coco supplementtion could increse postprndil insulin secretion nd, to greter extent, improve insulin resistnce. 3,31 Thus it is possile tht the hypocholesterolemic nd hypotriglyceridemic effect of CE is lso due to n increse in insulin secretion. Although LDL cholesterol levels did not seem to e ffected y CE tretment, totl cholesterol levels were normlised in dietic rts fter orl dministrtion of CE. Therefore it cn e suggested tht CE might possess hypocholesterolemic nd hypotriglyceridemic ctivity in STZ-induced dietic rts. The most common normlities in humns with poorly controlled type 1 or 2 dietes re hypertriglyceridemi nd low HDL cholesterol levels. 32 HDL is the smllest, densest lipoprotein with the lowest mount of triglyceride. Lower totl cholesterol nd higher HDL cholesterol levels represent very desirle iochemicl stte for prevention of therosclerosis nd ischemic conditions. 33 In its protective role ginst STZ ction, CE pretretment did not seem to prevent plsm HDL cholesterol levels from eing reduced y STZ, ut the levels were significntly enhnced (P <.5) fter 3 weeks of further tretment with CE. CONCLUSIONS The underlying mechnisms responsile for the lck of protective effect of CE on lipid profiles re not entirely understood nd still to e determined. This study indicted tht crude coco en extrct contining polyphenols nd other components might not hve protective effect ginst hypercholesterolemi, ut it does exert hypocholesterolemic effect in STZinduced dietic rts. ACKNOWLEDGEMENTS The uthors would like to cknowledge the finncil ssistnce provided y the Ministry of Science, Technology nd Innovtion of Mlysi (project IRPA EA1) nd the lortory fcilities of Universiti Putr Mlysi. 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