Effect of Prebiotics Inulin and Mannan on Lipid Profile and Intestinal Micro Flora of Hypercholesterolemic Rats

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1 J. Appl. Environ. Biol. Si., 6(8)22-31, , TextRod Pulition ISSN: Journl of Applied Environmentl nd Biologil Sienes Effet of Preiotis Inulin nd Mnnn on Lipid Profile nd Intestinl Miro Flor of Hyperholesterolemi Rts Ldn Aminlri 1, Seyed Shhrm Shekroroush 1 *, Seid Hosseinzdeh 1, Seed Nzifi 2, Jvd Sjedinfrd 3 1 Deprtment of Food Hygiene nd Puli Helth, Shool of Veterinry Mediine, Shirz University, Shirz, Irn. 2 Deprtment of Clinil Study, Shool of Veterinry Mediine, Shirz University, Shirz, Irn. 3 Deprtment of Physiology, Shool of Veterinry Mediine, Shirz University, Shirz, Irn. Reeived: April 3, 216 Aepted: June 25, 216 ABSTRACT The urrent study ws imed to determine the effet of Inulin nd mnnn on the lipid profile nd possile redution of serum holesterol level of rts indued hyperholesterolemi. Twenty-eight mle Wistr rts were divided into four experimentl groups s follow; ontrol group: fed stndrd ommeril diet; HC: stndrd diet ontining 1% holesterol nd 1% egg yolk powder; HC+M: HC+ 5% mnnn; HC+I: HC+ 5% inulin. During 5 dys, serum lipid profile, serum ALT nd AST, liver totl holesterol nd triglyeride, the ody nd orgn weights were mesured. HC+M nd HC+I groups showed signifintly lower holesterol, LDL holesterol, therogeni index nd serum AST thn HC group nd lso liver triglyeride nd totl holesterol were signifintly less in HC+M nd HC+I. HC+M nd HC+I diet hd signifintly ontrolled rts weight gin. These dt demonstrte tht mnnn nd inulin my e useful nd n e used s n lterntive or omplementry tretment in hyperholesterolemi nd relted disese onditions. KEY WORDS: Hyperholesterolemi; Inulin; Mnnn; Lipid profile INTRODUCTION Crdiovsulr diseses were onsidered s the mjor uses of dult s deth, worldwide. It hs een reported tht elevtion of ertin lood lipids levels re prinipl use of rdiovsulr disese nd oronry hert diseses [1]. Diets rih in sturted ft nd holesterol nd poor in fier, my result in elevted plsm holesterol levels [2]. Despite the long introdution of the dietry fier in 1953, the helth enefits of them were repetedly ddressed [3]. Preiotis, inluding dietry fiers nd fruto-oligoshrides, re inedile enefiilly food elements whih improving the host helth through the enhnement of growth nd tivity of ertin miro-flor of the host gstrointestinl trt[4, 5]. Preiotis hve n importnt role in diets euse of severl funtions, inluding: llowing growth nd lning of gut miro iot, ltering the omposition of the miro flor of gstrointestinl trts, ting s lterntive to proiotis, preventing some disese suh s dirrhe, hyperholesterolemi nd hypertension nd inresing immune system of the ody [4]. Inulin nd mnnn re lssified s insolule dietry fiers. Inulin is formed y the omposition of severl hins of frutose units linked y β (2 1) glyosidi onds frequently ended y gluose unit. Mnnnoligoshrides re nturlly present in the ell wlls of the yest Shromyes erevisie nd n e otined y entrifugtion of lysed yest ulture [6, 7]. It ws formerly hypothesized tht inrese in the formtion of suh fermented yproduts n potentilly redue the synthesis of hepti holesterol, moreover, disruption in the irultion of ile ids in the entero-hepti system nd lso fel emission of the extr ile slts re mong the mjor outomes of hypoholesterolemi [8, 9]. to explore the effet of feeding two kinds of preioti (inulin nd mnnn) on the lipid profile nd possile redution of serum holesterol level of rts indued hyperholesterolemi y dding high dose of holesterol nd egg yolk powder to their diet nd to study their improving intestinl miro flor y these preiotis. MATERIALS AND METHODS Experimentl nimls Twenty eight mle Wistr rts (2±1 grm) were rndomly divided into four groups (n=7). The nimls were mintined in entrl, smll niml house under ontrolled temperture t 22±2 C nd reltive humidity of 56% to 6% with 12-h light exposure in dily yle from 7.m. to 7 p.m. nd were fed on ommerilly diet (The * Corresponding uthor: S.S. Shekrforoush, Deprtment of Food Hygiene nd Puli Helth, Shool of Veterinry Mediine, Shirz University, Shirz, Irn. Fx: , e-mil: shekr@shirzu..ir 22

2 Aminlri et l.,216 stndrd pellet feedstuff ontined 17.9% protein, 48.5% strh, 4.9% sugr, 5.3% rude fier, 4.6% ft nd 7.1% sh,.72% lium,.6% phosphorus,.23% mgnesium nd.25% hloride). Animls were fed d liitum. All nimls were dpted for period of one week efore the experimentl session.the individul rt weight ws reorded weekly throughout the experiment. All over the experimentl proedures, the ethis of niml welfre ws pproved y Shirz University Animl Welfre Lws, Guidelines nd Poliies in Irn). Diet nd experimentl design The four experimentl group inluded ontrol: norml diet (ommeril diet); HC: high-holesterol diet (ommeril diet with 1% holesterol (Merk, Drmstdt, Germny) nd 1% egg yolk powder (Nrin ompny, Irn)); HC+I: high-holesterol diet+5% inulin (Sensus, Netherlnd); HC+M: high-holesterol diet+5% mnnn (Soren teh, Irn). After one week of dpttion the nimls were fed d liitum during 5 dys nd ody weight were reorded every week. Miroil nlysis of fel smples In order to the miroil nlysis of the nimls, fel smples were seprtely otined during 21 nd 5dys following onset of the trils whih ws promptly proessed within 1 h post olletion. The smples were susequently homogenized using stomher. The preprtions were then serilly diluted followed y suulture in triplite on plte ount gr (Merk, Drmstdt, Germny)for totl ount nd VRBD gr (Merk, Drmstdt, Germny) for Enteroteriee ount, nd inuted eroilly t 37 C for 48 h. Orl gluose tolerne test After the dietry dministrtion, t dys 28 nd 5, four nd three nimls were respetively deprived of the diets for 12 h in order to tke gluose solution (5g/kg) y intr gstri gvge. The orl gluose tolerne test ws then performed on the lood smples were tken from til vle. The lood gluose ws reorded t, 3, 6, 9, nd 12 min using rpid lood gluose meter (Au-hek ompny, USA)[1]. Smples olletion At the end of dys 28 nd 5 of the trils, the overnight-fsted rts were nesthetized y diethyl ether, the serum smples were then olleted from hert following entrifugtion t 3 g for minutes nd kept t -7 C for further nlysis. Liver, hert, spleen nd kidneys were removed, rinsed in hilled sline solution, lotted on filter pper, nd weighed seprtely. The orgns were kept t -2 C until nlysis. The nlysis of liver for determintion of totl holesterol nd triylglyerols ws finlly rried out. Serum nd liver lipids nlytil proedures Totl holesterol (TC), triglyeride (TG), nd high-density lipoprotein (HDL) holesterol of the ser were nlyzed y olorimetri retions using speifi enzymti kits (Biorex Frs, Irn).To nlyze other plsm lipid frtions inluding Low-density lipoprotein (LDL) holesterol, the Friedewld eqution ws employed to lulte the very lowdensity lipoprotein (VLDL) holesterol, Atherogeni index (AI), LDL/HDL rtio nd TC/HDL rtio [11]. Liver lipids were extrted nd purified ording to the method of Hr, et l. (1978) with few modifitions [12]. Briefly, 18 ml of hexne: isopropnol (3:2) ws dded to 1 grm of liver smple, the mixture ws homogenized(di 18B, IKA, Germny) t 5 gfor 3s nd mintined over night t 4 C. The mixture ws entrifuged t 5 g for 2 min. Non-lipid onstituents in the extrt were removed y mixing two time volumes of the superntnt with 12 ml of queous sodium sulfte (prepred from 1g of the nhydrous slt nd ml of wter). The hexne-rih lyer ws refully olleted the distint upper queous phse. The extrted TG nd holesterol were mesured using ommeril enzymti kits (Biorex Frs, Irn). Serum enzymti nlytil proedures The serum smples were nlyzed for liver enzymes ALT nd AST y olorimetri retions using speifi enzymti kits (Biorex Frs, Irn). Sttistil nlysis The dt of every three replites were nlyzed using SPSS (version 19.) softwre nd sujeted to one-wy nlysis vrine. The differene of mens etween groups ws lso nlyzed using Dunn s multiple omprison test. The level of P <.5 ws onsidered s signifint. 23

3 J. Appl. Environ. Biol. Si., 6(8)22-31, 216 RESULTS AND DISCUSION In the lipid-lowering potentil of dietry inulin nd mnnn in Wistr rts ws studied, here. There is lk of informtion to ompre the effet of inulin nd mnnn in lipid profile of hyperholesterolemi rts. Mnnn nd inulin re onsidered s dietry fier. The dietry fiers re likely fermentle y fel teri of the eum nd olon to produe short-hin ftty ids (ette, propionte nd utyrte) nd ltte [13]. The effets of dministering dietry fiers on serum lipid profile (serum totl triglyeride, holesterol, HDLholesterol, LDL-holesterol, VLDL-holesterol, therogeni index, HDL/LDL holesterol rtio nd holesterol/ HDL rtio) t the dys 28 nd dy 5 post exposure re shown in Fig.1. The sttistil nlysis of serum triglyerides (Fig. 1A) showed no signifine differene etween tretment groups. However serum triglyerides ws signifintly higher t dy 5, thn dy 28 (P<.5). On dy 28, tretment groups reeiving mnnn (HC+M) nd inulin (HC+I) showed signifintly lower holesterol thn HC group suh tht derese of serum holesterol y 33.9% nd 21.1%, respetively ws oserved. On dy 5, serum holesterol in HC group ws 3.1% higher thn ontrol group (P<.5) nd HC+M diet signifintly ontrolled serum holesterol nd ws similr to ontrol diet. Inulin showed oservle effet on serum holesterol, ut this effet did not pper to e signifint (Fig. 1B). These results showed tht mnnn hd more ontrolling effet thn inulin on dys 28 nd dy 5 of tretment. A signifint postprndil triglyeride lowering effet (27% to 61%) susequent to the orl dministrtion of 1 g oligofrutose /1g diet to mle rts fed high-ft diet ws reported y Delzenne nd Kok (1999). The effet ws likely used y reduing the tivity nd expression of ll lipogeni enzymes in the liver [14]. The hypothesis ws lter more lrified when it ws desried tht the min influenes re resulted from redution in the hepti de novo ftty id nd triylglyerol synthesis [8]. Gllher et l. (2) lso showed signifintly redution of serum nd liver holesterol of rts fed 1% gluomnnn nd hitosn. They suggested tht inresing the visosity of intestinl ontents used y gluomnnn feeding ws potentilly ssoited with lowering of holesterol sorption [13]. In this study HC diet used 51.9% nd 137.4% inrese in serum LDL-holesterol fter 28 nd 5 dys of tretment (P<.5). HC+M nd HC+I groups showed signifint deresing effet on LDL holesterol on dys 28 nd 5 (P<.5). Deresed LDL-holesterol on dy 28 for HC+M nd HC+I groups were 65.3 nd 59.%, respetively ompred to ontrol (P<.5). On dy 5 preioti diets signifintly ontrolled LDL-holesterol whih ws similr to ontrol diet. Our results lso showed no signifint differene for holesterol lowering effet of mnnn nd inulin diet (P>.5) (Fig. 1C). No signifint differene in the serum HDL-Cholesterol ws shown etween the experimentl groups on the dys 28 nd 5, exept for HC+I diet whih showed signifint inrese y 19.5% ompred to ontrol group on dy 28. Despite of hving high holesterol diets, rts hd higher serum HDL-holesterol on dy 5 thn dy 28 (Fig. 1D). Sttistil nlysis of serum VLDL-holesterol showed no signifint differene etween treted groups on oth dy 28 nd dy 5 (Fig.1E). It hs een hypothesized tht the prinipl mehnism for the lipid lowering tion of preiotis ws resulted from hnges in the hepti de novo lipogenesis, espeilly those ffeting VLDL prodution nd seretion, the hnges were preioti dose dependent []. On dy 28 of tretment therogeni index ws signifintly lower in rts fed HC+M nd HC+I diet y 42.4 nd 46.6%, respetively ompred with ontrols, wheres it ws signifintly greter in rts fed HC diet. There ws no signifint lowering effet etween HC+M nd HC+I diet on dy 28 ut HC+M hd signifintly more lowering effet of therogeni index on dy 5 (Fig.1F). Results of serum Cholesterol/HDL rtio re shown in Fig.1G whih showed signifint derese for oth HC+M nd HC+ I diet on dy 28. HC+I diet lso showed signifint deresing effet on dy 5. On the 28 th dysof the experiments signifint hnges in the serum LDL/HDL holesterol ws oserved mong ll the trils. HC diet signifintly inresed LDL/HDL holesterol rtio y 56.8% ompred to ontrol wheres HC+M nd HC+I diet ws signifintly deresed this rtio y 64.9% nd 64.7% nd there were no signifint different etween HC+M nd HC+I diet. On dy 5 lso HC+M nd HC+I diet signifintly deresed LDL/HDL holesterol rtio ompred to HC group (Fig.1H). 24

4 Aminlri et l.,216 A Serum triglyeride (mg/dl) B Serum holesterol (mg/dl) , 28 5 Tretment dy 28 Tretment dy 5 C Serum Ldl (mg/dl) D Serum Hdl (mg/dl) Tretment dy 28 5 Trerment dy E Serum Vldl (mg/dl) F Atherogeni index s Tretment dy Tretment dy G Cholesterol/Hdl rtio , H Ldl/Hdl rtio Tretment dy Fig. 1. The effet of supplementtion of diet with preiotis mnnn nd inulin on A: serum triglyeride, B: totl holesterol, C: LDL- holesterol, D: HDL-holesterol, E: VLDL-holesterol, F: therogeni index, G: Cholesterol/ HDL rtio nd H: LDL/HDL rtio in hyperholesterolemimle Wistr rts. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Brs represent stndrd devition vlues. Different letters in the sme smpling dy indite signifint differenes t the level of.5. The tivities of ALT nd AST were mesured to ess heptoellulr dmge [16]. Serum ALT nd AST re shown in the Fig.2. No signifint differene in serum ALT tivity of ll groups ws seen on dy 28 ut it signifintly deresed in rts fed HC+I diet on dy 5 y 38.8% ompred to HC diet nd ws lose to ontrol group (Fig.2A).HC diet lso hd signifint inresing effet on serum AST on dys 28 nd 5 y 2.19% nd 16.38% respetively, whih serum AST in mnnn nd inulin group ws similr to ontrol group on dy 28 nd even signifintly less in dy 5 (Fig.2B).These oservtions led to the onlusion tht high holesterol diet n indue hepti dmge resulting in elevted hepti enzymes nd tht preiotis n llevite these hnges. These results Tretment dy 25

5 J. Appl. Environ. Biol. Si., 6(8)22-31, 216 were supported y El-Mhmoudy et l.(214)who investigted the effet of mnnn-oligoshride on the lipogrm in hyperlipidemi rts [16]. A ,, Alt (IU/L) Tretment dy B Ast (IU/L) Tretment dy Fig. 2. The effet of supplementtion of diet with preiotis mnnn nd inulin on A: serum ALT nd B: serum AST in hyperholesterolemimle Wistr rts. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Brs represent stndrd devition vlues. Different letters in the sme smpling dy indite signifint differenes t the level of.5. HC diet inresed liver triglyeride signifintly (P<.5). Sttistil nlysis for the effet of feeding different preioti on liver totl triglyeride reveled tht there ws higher signifint deresing effet on dy 28 nd 5 y 21.5% nd 31.1% for HC+M diet nd 2.9% nd 3.1% for HC+I diet respetively thn did the orresponding HC diet (P<.5) (Fig. 3A). HC diet inresed liver totl holesterol signifintly(p<.5). Effet of dietry mnnn nd inulin on liver totl holesterol showed the signifint derese on dys 28 nd 5 ompred to HC nd lso ontrol group (Fig. 3B). Our results were lso supported y Delzenne et l. (1999) whih explined dietry supplementtion with oligofrutose (1g/kg), dereses serum triylglyerols in serum nd VLDL of rts. It lso showed signifintly derese in triylglyerol nd phospholipid onentrtions in oth lood nd liver [14]. Gllher (2) lso showed signifintly redution of totl liver holesterol y dding 1% of gluomnnn nd hitosn tohyperholesterolemi rts [13]. 26

6 Aminlri et l.,216 A 25 2 Liver tryglyeride (mg/dl) 1 d d 5 B Tretment dy 2 Liver holesterol (mg/dl) 1 d Tretment dy Fig. 3. The effet of supplementtion of diet with preiotis mnnn nd inulin on A: liver triylglyeride nd B: liver totl holesterol in hyperholesterolemimle Wistr rts. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Brs represent stndrd devition vlues. Different letters in the sme smpling dy indite signifint differenes t the level of.5. The effets of the dietry tretments on the fel totl ount nd Enteroteriee ount re presented in Fig. 4. After 21 nd 5 dys of tretment, fel totl ount of HC group ws signifintly lower ompred to the other groups (P<.5). However, totl ount in HC+M nd HC+I groups were similr or even more thn ontrol group (Fig.4A). It is possile tht high onentrtion of holesterol in the diet of HC group hs prevented growth of some speies of teri in the lrge intestine of rts. However, in the HC+M nd HC+I groups; it ppers tht the stimulting effet of preiotis on teril growth hs indeed overome the inhiitory influene of holesterol. HC, HC+I nd HC+M diets signifintly inresed fel Enteroteriee ount ompred to ontrol diet fter 21 dys (P<.5). However, no signifint hnges were oserved etween ll the groups over the 5 dys (Fig. 4B).Our results support the onept of seletive stimultion of fees teri y preiotis [4, 5, 17]. 27

7 J. Appl. Environ. Biol. Si., 6(8)22-31, 216 A 12 Totl ount (Log fu/g) Tretment dy B 8 Enteroteriee (Log fu/g) Tretment dy Fig. 4. The effet of supplementtion of diet with preiotis mnnn nd inulin on A: Totl ount nd B: Enteroteriee ount of fees in hyperholesterolemimle Wistr rts. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Brs represent stndrd devition vlues. Different letters in the sme smpling dy indite signifint differenes t the level of.5. Weight gin for HC group ws signifintly more on dys 35, 42 nd 5 nd HC+M nd HC+I diet hd signifintly ontrolled their weight gin ompre to HC diet(fig.5). The weight gins during the study period were respetively 121.g nd 144.4g in the ontrol nd the HC diet groups. However, when feeding the HC+M nd HC+I diet, the weight gin ws 95.5g nd 15.g, respetively. Gllher et l. (2) showed weight gin redution of rts when fed 1% gluomnnn nd hitosn ompred to ontrol diet nd explined tht the lower dily food intke of these groups re the reson of weight ontrol [13].A reltive lower weight gin in the preioti ingested group ws proly ssoited with the presene of lrge mounts of the insolule fiers in the GIT. This phenomenon ws previously reported y erlier studies tht diets ontining high insolule fiers produing less metolize energy [18]. 28

8 Aminlri et l., * * weight (g) * Tretment dy Fig. 5. The effet of supplementtion of diet with preiotis mnnn nd inulin on weight of hyperholesterolemimle Wistr rts during 5 dys tretment. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Mens with strs in eh dy of tretment re signifint t.5 levels. The Effet of supplementing different preiotis on weights of liver, kidney, hert nd spleen of rts re shown in Fig.6. It ws found tht there ws no signifint inrese in liver weight in HC group ompred to ontrol fter 28 dys of feeding. In dy 5 of tretment, HC group showed inresed liver weight ompred to the ontrol (P<.5) ut when feeding the HC+M nd HC+I diet, they hd no differene with the ontrol group (Fig.6A). HC diet indued signifint weight inrese in the kidneys when it ws ompred to the ontrol group t the 28 th dy (P <.5). On dy 5 of tretment, kidneys weight of tretment groups were lose to ontrol group(fig.6b).on dy 28 nd 5 hert weight of rts in ll groups ws similr (Fig.6C).On dy 28 f experiment spleen weight of HC group ws similr to tht of ontrol. However, on dy 5 HC diet hd signifintly inresed the spleen weight (P <.5), while this prmeter in HC+M nd HC+I groups ws similr to tht of ontrol (Fig.6D). These results showed tht dietry inulin nd mnnn t dose of 5%were well tolerted y the rts nd did not use over growth nd inrese in the weight of orgn nd lso redued the dverse effets of holesterol in the ody weight gin. A Liver weight (g) ,,, B Kidney weight (g) Tretment dy Tretment dy 29

9 J. Appl. Environ. Biol. Si., 6(8)22-31, 216 C Herth weight (g) D Spleen weight (g) ,, Tretment dy Tretment dy Fig. 6. The effet of supplementtion of diet with preiotis mnnn nd inulin on A: liver, B: kidney, C: Hert nd D: spleen weight of hyperholesterolemimle Wistr rts. Control ( ), High holesterol ( ), High holesterol + mnnn ( ), High holesterol + inulin ( ). Brs represent stndrd devition vlues. Different letters in the sme smpling dy indite signifint differenes t the level of.5. Tle1shows the results of gluose tolerne test in rts whih ws performed efore srifiing. In dy 28 nd 5, t zero time, lood gluose of HC, HC+M nd HC+I groups whih ws signifint ompred to the ontrol (P <.5). Additionlly, no signifint differene mong groups following 12min post-exposure ws oserved. Sttistil nlysis for the effet of feeding different preioti on lood gluose t time zero indites signifint inrese in HC, HC+M nd HC+I groups ompre to ontrol group. At time 6 HC diet signifintly inresed lood gluose ut there were no different etween groups in time 3 nd 9 nd 12. A derese in gluose levels ws intended susequent to the dministrtion of mnnn nd inulin sine mny reserhers hve previously hypothesized the role of fruto-oligoshrides in the regultion of gluose. For exmple Delzenne nd Kok (1999) hd oserved tht hving fruto-oligoshrides, whih re plying role in the nutritionl regultion of lipogenesis, signifintly redues serum insulin nd gluose [14]. However, our results were omprle to the study of Cusey et l. (2) on humns who found lose ssoition etween n inrese in the levels of insulin nd glugon levels following onsumption of insolule fiers [19]. Tle 1. The effet of supplementtion of diet with preiotis mnnn nd inulin on serum gluose (mg/ dl) of hyperholesterolemimle Wistr rts given gluose solution (5 g/kg). Time [min] Dy Tretment groups Control 82.5±2.3 A 184.5±19.1 B ±1.6 B 161.±8.7 B 127.±8.8 C (n=4) High holesterol 19.±4.6 A 3.±3.6 B ±8.8 B 3.33±13.9 B 145.±21.9 AB High holesterol+ mnnn ±3.1 A 16.±7.8 B 167.±7.2 B 7.±7.3 B ±2.5 A High holesterol+ inulin 99.5±1.3 A 17.75±1.3 B ±8.3 B 165.±3.2 B ±9.1 C 5 (n=3) Control 93.33±5.8 A ±1.7 B 2.67±8.2 B ±6. B 14.33±7. B High holesterol 11.±4.5 A 162.±9.2 B ±7.5 B 18.67±14.5 B 172.±11.9 B High holesterol+ mnnn 13.67±3.8 A ±2.7 A 143.±5.6 A 141.±6.7 A ±6.27 A High holesterol+ inulin 129.±1. A 8.±5.9 AB ±11. AB 16.67±.6 AB ±1.5 B The different pitl letters indite signifint differenes in row(p <.5).The different smll letters in the sme olumn within the sme dy indite signifint differenes (P <.5). In onlusion, our results suggest the impertive role of inulin nd mnnn to redue the serum levels of holesterol, LDL nd VLDL. It ould e suggested tht the preioti my e useful nd n e used s n lterntive or omplementry tretment in hyperlipidemi nd relted disese onditions. 3

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