Veterinry Worl, EISSN: 2231-0916 Aville t www.veterinryworl.org/vol.10/june-2017/22.pf RESEARCH ARTICLE Open Aess Honey n repiring mge of liver tissue ue to protein energy mlnutrition through inution of enogenous stem ells R. Heru Prsetyo 1 n Ek Prmyrth Hestinh 2 1. Deprtment of Prsitology, Fulty of Meiine, Universits Airlngg, Sury, Est Jv, Inonesi; 2. Deprtment of Veterinry Antomy, Fulty of Veterinry Meiine, Universits Airlngg, Sury, Est Jv, Inonesi. Corresponing Author: R. Heru Prsetyo, e-mil: rheru_prsetyo@yhoo.o.i Co-uthor: EPH: ekmirt@gmil.om Reeive: 26-10-2016, Aepte: 08-05-2017, Pulishe online: 29-06-2017 oi: 10.14202/vetworl.2017.711-715 How to ite this rtile: Prsetyo RH, Hestinh EP (2017) Honey n repiring mge of liver tissue ue to protein energy mlnutrition through inution of enogenous stem ells, Veterinry Worl, 10(6): 711-715. Astrt Aim: This stuy ws to evlute effet of honey in repiring mge of liver tissue ue to energy protein mlnutrition n in moiliztion of enogenous stem ells. Mterils n Methos: Mle mie moel of egenertive liver ws otine through foo fsting ut still hve rinking wter for 5 ys. It use energy protein mlnutrition n mge of liver tissue. The ministrtion of 50% (v/v) honey ws performe for 10 onseutive ys, while the positive ontrol group ws fste n not given honey n the negtive ontrol not fste n without honey. Oservtions of regenertion the liver tissue se on histologilly exmintion, oservtion of Hsp70 expression, n homing signl se on vsulr enothelil growth ftor-1 (VEGF-1) expression using immunohistohemistry tehnique. Oservtion on expression of CD34 n CD45 s the mrker of uto moiliztion of hemtopoieti stem ells using flow ytometry tehnique. Results: There is regenertion of the liver tissue ue to protein energy mlnutrition, erese of Hsp70 expression, inrese of VEGF-1 expression, n high expression of CD34 n CD45. Conlusion: Honey n improve the liver tissue se on: (1) Moiliztion of enogenous stem ells (CD34 n CD45); (2) Hsp70 n VEGF-1 expressions s regenertion mrker of improvement, n (3) regenertion histologilly of liver tissue Keywors: enogenous stem ells, honey, liver tissue, protein energy mlnutrition, regenertion. Introution Nutrition efiieny ws n helth prolem, espeilly in evelopment ountries, inlue Inonesi. Sine 2000 in Inonesi showe inrese iniene of protein energy mlnutrition (PEM) [1]. FAO mention tht until 2016, more thn 20 million Inonesin popultion is still unernourishe [2]. More thn 50% of eths of mlnourishe hilren ue to lk of energy protein whih inreses the numer of eths ue to intestinl isorers n irrhe [1]. In generl, PEM is the most frequent use of quire immune efiieny [3] whih n le to opportunisti infetion y intestinl prsites with symptoms of hroni irrhe n tht will inrese mortlity [4]. From previous stuies, the mie were onitione PEM in ition to otine n inrese expression of Hsp70, erese expression of prostglnin E2, n expression of immunogloulin A were exmine immunohistohemilly (IHC), lso histologilly visile mge to the epithelium of the intestinl muos n ours villus trophy, whih Copyright: Prsetyo n Hestinh Open Aess. This rtile is istriute uner the terms of the Cretive Commons Attriution 4.0 Interntionl Liense (http://retiveommons.org/lienses/ y/4.0/), whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver (http:// retiveommons.org/puliomin/zero/1.0/) pplies to the t me ville in this rtile, unless otherwise stte. will le to erese in the funtion of sorption [5] the impt reue nutritionl intke n orgns mge, inluing liver mge. This stuy ime to evlute the impt of honey to repir liver tissue n its effet on moiliztion of enogenous stem ells in the liver tissue mge ue to PEM. The results of this stuy re expete s input in etermining poliies for hnling mlnourishe. Mterils n Methos Ethil Approvl The present stuy ws pprove y ethil ommittee vie Ethil Clerne No: 065- KE (Komisi Etik Penelitin, Fkults Keoktern Hewn, Universits Airlngg, Animl Cre n Use Committee (ACUC)). PEM moeling uses liver egenertion This stuy ws strte y PEM s the uses of liver egenertion of mle mie. PEM ws one y stning wter every 8 hours for the mle mie without ny foo in the ourse of 5 ys [6,7]. Fsting ws one in 5 ys euse in the 5 th y the liver unergoes egenertion n trophy of the intestinl epithelil hs een mge [8]. This stuy use mle Bl/ mie 8-10 weeks ol, 20-30 g oy weight in goo onition [9]. Mie otine from lortory nimls experiments in Fulty of Veterinry Meiine, Universits Airlngg. Mie kept in plsti ge spe per Veterinry Worl, EISSN: 2231-0916 711
Aville t www.veterinryworl.org/vol.10/june-2017/22.pf iniviul in lortory nimls experiments in Fulty of Veterinry Meiine, Universits Airlngg. Reserh Design The reserh is n experimentl stuy with esign the rnomize post-test only ontrol group esign. The stuy ws ivie into 4 groups of 10 mies eh (se formul (t-1)(n-1)>15). t (tretments)=4 groups n n (replites)=6 (miniml). Desription of tretment: 1. The negtive ontrol group (T1): Mle mies not fste n without honey. 2. The positive ontrol group (T2): Mle mies were fste for 5 ys n without honey. 3. The tretment group (T3): Mle mies not fste for 5 ys, then given 50% (v/v) honey in the rinking wter for the next 5 ys fter fste. 4. The tretment group 4 (T4): Mle mies were fste, then given 50% (v/v) honey in the rinking wter for the next 5 ys fter fste. Honey tht use in this stuy ws rw multiflor honey, Apis orst prout from Btu Mlng, Est Jv, Inonesi. After the tretment, whole loo is tken from the hert n is ollete in heprin tue, then performe euthnsi y ervil islotion n follow y mking the liver orgn. The liver orgn put in pot tht lrey ontins 10% formlin solution fter lensing with sline. Oservtions were tken for regenertion of liver tissue use histologilly tehnique, Hsp70 expression n vsulr enothelil growth ftor-1 (VEGF- 1) expression use IHC tehnique, n the perentge of CD34 n CD45 use flow ytometry tehnique. Oservtion of hemtopoieti stem ells moiliztion s enogenous stem ells se on expression of CD34 n CD45 with flow ytometry metho After the mie were trete, further exmintion of whole loo smple is tken through ri punture n inserte into the tue ws ontent heprin to prevent ogultion. Oservtions on the expression of CD34 n CD45 were nlyze y flow ytometry Flow ytometry metho, strting with the preprtion of whole loo entrifugtion in temperture of 4 C, with spee of 6000 rpm for 15 min. Results entrifuging the ell in the form of sluge mixe with ytoperm mount of 2 times the numer of ells re nlyze. A mixture of ells n ytoperm ws entrifuge to otin superntnt n pellet. Wshing the pellet mount 4 times of otine ell numer from the first entrifugtion. Furthermore, lysis uffer mount of 2 times of the first otine ell numer. After tht lele ntioy onjugte to eh smple, five tues re prepre n proesse in prllel. (1) Single stining with CD34 PE e to the wsh tue. (2) Doule stining with CD34 PE n CD45 PerCP n CD105 FITC wsh tue. (3) Doule stining with CD34 PE n CD45 PerCP truount tue. The entire smple ws then store t 4 C in the rk n nlyze using flow ytometry for 1 h [10]. IHC tehnique for oservtion of Hsp70 n VEGF-1 expression IHC oservtion ws performe to etermine the expression of Hsp70 s mrker of improvement n VEGF-1 s mrker of growth ftor. Before to IHC methos were me histologil preprtion, y wy of n inision is me trnsversely liver tissue from prffin loks. Further exmintion y mking outwr through IHC tehniques using monolonl ntioies nti-hsp70 n monolonl ntioies nti VEGF-1. Oservtions of Hsp70 n VEGF-1 expression were me using light mirosope with mgnifition of 200 times, n the expression of eh vrile is inite y the numer of ells with rownish isolortion hromogen in eh inision [11]. Histologilly tehnique Histologilly exmintion se on oservtion of liver tissue regenertion. Histologilly exmintion egins with the mking of histologil preprtions, suh s the following: Mie liver fixtion in 10% uffer formlin. Susequently, liver ehyrte in lohol solution with higher onentrtion, i.e., from 70%, 80%, 90%, n 96% (solute). Then o the lering in the liver of mie in xylol solution. Furthermore, performe emeing using liqui prffin n mie liver were put into mols ontining liqui prffin. Before stine n setioning performe, n inision using mirotome n mounte on glss ojets. Furthermore is one the stining y removing of prffin with xylol then put into solution of lohol with erese onentrtion n then put into stin mtter. The lst stge fter stine is one mounting, put into wter or lohol to remove exess stin. Then put into solution of lohol with inresing onentrtion, n then put into xylol. Preprtions then overe with over glss n mounte with entelln. Mirosopi exmintion is one with light mirosope with mgnifition 200 [12,13]. Sttistil nlysis Expressions of mie liver, VEGF-1, n Hsp70 were sttistilly nlyze using SPSS 15 for Winows XP with the level of signifine 0.05 (p=0.05) n the onfiene level 99% (α=0.01). Steps of omprtive hypothesis tests re s follows: Test t normlity with the Kolmogorov Smirnov test, homogeneity of vrine test, nlysis of vrine ftoril, post ho test (lest signifint ifferene test) using the Tukey highly signifint ifferene 5%. Results n Disussion Dt were ollete from 32 mle mies were ivie into four tretments: Negtive ontrol group (T1) is norml liver without honey; positive ontrol group (T2) is egenertive liver without honey; (T3) group is norml liver + 50% (v/v) honey in rinking Veterinry Worl, EISSN: 2231-0916 712
Aville t www.veterinryworl.org/vol.10/june-2017/22.pf wter for 5 ys; (T4) group is egenertive liver + 50% (v/v) honey in rinking wter for 5 ys. In etil, the results of the stuy re s follows: The effetively of honey ws se on: (1) moiliztion of enogenous stem ells (CD34 n CD45); (2) Hsp70 n VEGF-1 expressions s regenertion Hsp70 s mrker of improvement [8] n VEGF-1 s mrker of growth ftor [14]; n (3) regenertion histologilly of liver tissue explin, why HSP70 n VEGF-1 re heke s mrkers. Moiliztion of enogenous stem ells nlyze y flow ytometry se on inrese onentrtion of CD34 n CD45. The nlysis shows tht: Either negtive ontrol group (T1), positive ontrol group (T2) or group (T3) showe no moiliztion of enogenous stem ells, se on the perentge of CD34 n CD45 whih re t perentge of <20% (Figure-1-), wheres in the group (T4) showe moiliztion of enogenous stem ells is se on the perentge of CD34 n CD45 whih re lote on the perentge of over 70% (Figure-1). Bse on sttistil lultions T2 groups ws signifintly ifferent (p<0.05) thn the other three tretments (T1, T2, n T3), wheres mong the three tretments no signifint ifferene (p>0.05) (Figure-1). The proess of moiliztion n our in severl wys, mong others re the immune responses or inflmmtory retion ue to injury signls (ytokines, nuler ftor kpp B, β tenin through Wnt) from the tissue mge n homing signls like VEGF-1 tht ppers if there re n inflmmtory retion. Homing signl tht ours plys role in the reruitment of stem ells s well s in ells unergoing poptosis [15-17]. Hsp70 expression in this stuy showe erese in the group reeiving honey, either in group T3 (0.375± 0.43 ) n T4 group (1.25± 0.09 ). Derese expression of Hsp70 fter the ition of honey on T3 groups ws not signifint (p>0.05) with T1 group (0.125± 0.90 ). In the T4 group espite the ownturn ut signifintly iffer signifintly with T1 n T3 groups (p<0.05). This inites tht the honey tretment, on moleulr sis is not optiml. In the T2 group (2.875±0.46 ) n inrese in exessive n signifintly ifferent when ompre with the other 3 groups. This shows liver tissue mge is experiening PEM n not e given honey (Figure-2). Derese of Hsp70 expression inites poptosis inhiition n regenertion of liver tissue [18]. The inrese of growth ftor se on VEGF-1 expression, in the norml ontrol group (T1) ws on the sore 0.25± 0.60 (VEGF-1 expression etween 1% n 5%). The group of liver egenertive (T2) ws on the sore 1.125± 0.78 (VEGF-1 expression etween 6% n 25%). The group use 50% (v/v) ee-honey (T4) ws on the sore 2.75±0.44 (VEGF expression >50%) (Figure-3). This shows n improvement of liver tissue through proess of homing signls [19]. Furthermore, effetiveness of honey in this stuy se on the regenertion of the liver. The regenertion Figure-1: Flow ytometry nlysis of enogenous stem ells moiliztion. () The ontrol negtive group (T1): Expression of CD34 n CD45 of 18.25±1.26. () The ontrol positive group (T2): Expression of CD34 n CD45 of 18.12±1.25. () The group 3 (T3): Expression of CD34 n CD45 of 18.5±1.27. () The group 4 (T4): expression of CD34 n CD45 of 74.12±1.87. The ifferent supersripts inite signifint ifferene t p<0.05. Figure-2: Hsp70 Expression in mie liver tissue using immunohistohemilly tehnique on severl tretments. () The ontrol negtive group (T1), sore Hsp70 expression=0.125± 0.90 ; () The group 3 (T3), sore Hsp70 expression=1.25± 0.09. () The ontrol positive group (T2), sore Hsp70 expression=0.375± 0.43. () The group 4 (T4), sore Hsp70 expression=2.875±0.46. The ifferent supersripts inite signifint ifferene t p<0.05. n e oserve through the metho of histopthology ntomy with hemtoxylin n eosin (H n E) stining. Mirosopi exmintion showe tht the group of 50% (v/v) honey (T4), leing to the ourrene of liver tissue repir, se on heptoytes expression n proution of oth mirovesiulr n mrovesiulr vuoltion (Figure-4). The ontrol negtive group (T1) with norml liver hs norml heptoytes expression, hve unnt ler ytoplsm whih hs Veterinry Worl, EISSN: 2231-0916 713
Aville t www.veterinryworl.org/vol.10/june-2017/22.pf Figure-3: Vsulr enothelil growth ftor-1 (VEGF-1) expression in mie liver tissue using immunohistohemilly tehnique on severl tretments. () The ontrol negtive group (T1), sore VEGF-1 expression=0.25± 0.60 ; () The group 3 (T3), sore VEGF-1 expression=0.375± 0.43. () The ontrol positive group (T2), sore VEGF-1 expression=1.125± 0.78. () The group 4 (T4), sore VEGF-1 expression=2.75 ±0.44. The ifferent supersripts inite signifint ifferene t p<0.05. Figure-4: The regenertion of mie liver tissue using the histologilly tehnique with hemtoxylin n eosin stining in few tretments. () The ontrol negtive group (T1) ws showe norml heptoytes expression ( ); hve unnt ler ytoplsm whih hs fethery pperne ue to the presene of glyogen. (). The ontrol positive group (T2), ongestion of liver ( ) n hemorrhgi ( ), lso visile hemosierin ( ) ue to loo ell lysis (rownish yellow olor) with eposition of firin ( ) initing tht hroni ongestive hs ourre. Heptoytes with onense rkly stining ytoplsm euse eplete of glyogen. (). The group 3 (T3), liver oes not regenerte, it ppers there is ongestion ( ) n eposition of firin ( ). () The group 4 (T4), liver egin to regenerte with heptoytes expression whih proution of oth mirovesiulr n mrovesiulr vuoltion ( ), lthough it still looks little ongestion in loo vessel ( ). fethery pperne ue to the presene of glyogen (Figure-4). The ontrol positive group (T2), ongestion of liver n hemorrhge, lso visile hemosierin ue to loo ell lysis (rownish yellow olor) with eposition of firin initing tht hroni ongestive hs ourre. Heptoytes with onense rkly stining ytoplsm euse eplete of glyogen (Figure-4) Not the improvement in the form of liver oes not regenerte, it ppers there is ongestion, hemorrhgi n hemosierin expression (Figure-4). The group of liver egenertive (T1), liver ws ongeste extensive n hemorrhgi, lso visile hemosierin (yellow-rown) ue to loo ell lysis with firin eposition (y olor pink) initing tht hroni ongestion hs ourre (Figure-4). These results inite tht ministrtion of honey with ose of 50% (v/v) in rinking wter for 5 ys ws le to inue enogenous stem ells to migrte to where the efet. Repir egins on intestine orgn, mking it esier for the sorption of honey onsume. This ultimtely le to the proess of repir of liver tissue. Improvements to the liver n e seen in the results of histopthologil exmintion of the ntomy of the liver tissue with H n E stining. PEM in this stuy lso use firosis in liver tissue were experiening efet. Aoring to inue stem ells hve the ility s ntifiroti so tht honey therpy n e use in the tretment of tissue firosis relte to the hroni inflmmtory onition ue to PEM [20,21]. Furthermore, trigger of VEGF-1 whih ining to VEGF reeptor-1 (VEGFR-1) n ourre. VEGF-1 is homoimeri glyoproteins with moleulr weight of 45 kd. VEGF-1 is expresse y mny types of ells s homing signl. VEGF is omponent of the extrellulr mtri from stem ells hs role in supporting onuive miroenvironment for stem ells. Trigger presene of VEGF-1 - VEGFR-1 will pss series of signling tht tivtes stem ells ftor (SCF) interstitil. SCF or lso known s steel ftor is signling protein mehni on nihe so tht physiologilly ommunition will our [22,23]. Tretment of 50% (v/v) honey for 5 ys in mle mie with egenerte liver tissue n show: (1) Moiliztion of enogenous stem ells, in the form of mrkers CD34 n CD45 re expresse in loo serum; (2) inue homing signl in the form of inrese of growth ftor (VEGF-1) y IHC in liver tissue; (3) regenertion of liver tissue, heptoytes expression n proution of oth mirovesiulr n mrovesiulr vuoltion, lthough there is still little ongestion ut hemosierin expression n firin eposition hs not looke k. The results show tht the woun heling properties of honey inlue stimultion of tissue growth n enhne epitheliliztion. These effets re srie to nutritionl n ntioxint ontents n stimultion of immunity [24-26]. Conlusion Honey n e improvement the liver tissue se on: (1) Moiliztion of enogenous stem ells (CD34 n CD45); (2) Hsp70 n VEGF-1 expressions s regenertion mrker of improvement, n (3) regenertion histologilly of liver tissue. Veterinry Worl, EISSN: 2231-0916 714
Authors Contriutions Aville t www.veterinryworl.org/vol.10/june-2017/22.pf RHP: Reserh oorintor, prepre PEM moeling uses liver egenertion in mle mie, n ministrtion of 50% (v/v) honey in the rinking wter for the next 5 ys fter fste n revise the mnusript. EPH: Metho of histopthology ntomy n sttistil nlysis. All uthors re n pprove the finl mnusript. Aknowlegments The stuy ws supporte y funing from the Diretorte Generl of Higher Eution (DIKTI) 2016 (grnt no.018/sp2h/lt/drpm/ii/2016. 17 Fe 2016), The Ntionl Eution Ministry, Repuli of Inonesi. Competing Interests We elre tht they hve no ompeting interest. Referenes 1. Blk, R.E., Vitor, C.G., Wlker, S.P., Bhutt, Z.A., Christin, P., e Onis, M., Ezzti, M., MGregor, S.G., Ktz, J., Mrtorell, R. n Uuy, R. (2013) Mternl n hil unernutrition n overweight in low-inome n mile-inome ountries. Lnet, 382(9890): 427-451. 2. Aullh, A., Hort, K., Butu, Y. n Simpson, L. (2016) Risk ftors ssoite with neontl eths: A mthe se-ontrol stuy in Inonesi. Glo. Helth Ation, 9: 30445. 3. Chinen, J. n Shere, W.T. (2010) Seonry immunoeffiienies, inluing HIV infetion. J. Allergy Clin. Immunol., 125(2): 195-203. 4. Mrkell, E.K., John, D.T. n Krotoski, W.A. (1999) Mrkell n Voges Meil Prsitology. 8 th e. WB Suners Compny, St Louis. p25-37. 5. Prsetyo, R.H. (2010) Chnges in the Expression of CD4, IgA, PGE2, n Hsp70 Intestinl Muos Mus musulus Bl/ Protein Energy Mlnutrition Infete y Cryptosporiium. Disserttion, University of Airlngg, Inonesin. p37-45. 6. Chnr, R.K. (2003) Protein-energy mlnutrition n immunologil responses. J. Nutr., 21(5): 242-247. 7. Sntos, M.A., Ros, R. n Brieri, C. (1997) Effet of protein mlnutrition on the glyolyti n glutminolyti enzyme tivity on rt thymus n mesenteri lymph noes. Brz. J. Me. Biol. Res., 30(6): 719-722. 8. Prsetyo, R.H. n Sfitri, E. (2016) Effets of honey to moilize enogenous stem ells in efforts intestinl n ovrin tissue regenertion in rts with protein energy mlnutrition. Asin P. J. Repro., 5(3): 198-203. 9. Sfitri, E., Utm, S., Wiiytno, T.V., Snhik, W. n Prsetyo, R.H. (2016) Auto-regenertion of mie testile seminiferous tuules ue to mlnutrition se on stem ells moliztion using honey. Asin P. J. Repro., 5(1): ******** 31-34. 10. Mey, M.G. (2007) Flow Cytometry, Priniple n Aplitions. 1 st e. Humn Press, TOtow. p1-31. 11. Kumr, G.L. n Ruek L. (2009) Immunohistohemil Stining Methos. 5 th e. Dko North Ameri, Crpinteri, Cliforni. p11-14. 12. Crosy, K., Simeninger, J., Grnge, C., Ferrnte, M., Bernier, T. n Stnen, C. (2016) Immunohistohemistry protool for prffin-emee tissue setion-vertisement. Cell Signl. Tehnol., Aville from http://www. origene.om/support. Aesse on 03-08-2016. 13. Sumore, C.L. (2014) A Prtil Guie to Histology of the Mouse. 1 st e. John Willey & Son, Willey Blkwell, Chihester. p56-58. 14. Sfitri, E., Wiiytno, T.V. n Prsetyo, R.H. (2016) Honeyee prout therpeuti s stem ells homing for ovry filure. Vet. Worl, 9(11): 1324-1330. 15. Ross, M.H. n Pwlin, W. (2011) Histology Text n Atls. 6 th e. Lippinott Willim & Wilkins, Bltimore. p75-81. 16. Rntm, F.A., Ferinsyh, N. n Purwti, A. (2009) Stem Cell Explortion: Methos of Isoltion n Culture. 1 st e. Airlngg University Press, Sury. p25-39. 17. Rntm, F.A., Ferinsyh, N. n Purwti, A. (2014) Stem Cell Mesenhyml, Hemtopoetik n Moel Apliksi. 2 n e. Airlngg University Press, Sury. p45-50, 145-155. 18. Hermnn, M., Vrrier, S. n Muro, A. (2015) Strtegies to stimulte moiliztion n homing of enogenous stem n progenitor ells for one tissue repir. Front. Bioeng. Biotehnol., 2(3): 79. 19. Brker, N. (2014) Ault intestinl stem ells: Critil rivers of epithelil homeostsis n regenertion. Nt. Rev. Mol. Cell Biol., 10(5): 19-33. 20. Dussut, C., Brunet, J.L., Higes, M., Colourne, J.K., Lopez, J., Choi, J.H., Mrtín-Hernánez, R., Botís, C., Cousin, M., MDonnell, C., Bonnet, M., Belzunes, L.P., Moritz, R.F.A., Conte, Y.L. n Alux, C. (2012) Gut pthology n responses to the mirosporiium nosemerne in the honey ee pis mellifer. PLoS One, 7(5): 1-12. 21. Hu, C. n Li, L. (2015) Review: In vitro ulture of isolte primry heptoytes n stem ell-erive heptoyte-like ells for liver regenertion. Protein Cell, 6(8): 562-574. 22. Niuni, M., Azimi, E., Shirvi, A. n Nzri, Z. (2012) Honey Bee Venom will Differentite Mesenhyml Stem Cells in to the Osteoyte. Interntionl Conferene on Applie Life Sienes. Turkey. Septemer; 2012. p247-250. 23. Dennis, E.A. n Brshw, R.A. (2011) Interellulr Signling in Development n Disese. 1 st e. Elseiver In., Sn Diego. p121-122. 24. Al-Wili, N., Slom, K. n Ghmi, A.A. (2011) Honey for woun heling, ulers, n urns; t supporting its use in linil prtie. Si. Worl J., 5(11): 766-787. 25. Lusy, P.E., Coomes, A. n Wilkinson, J.M. (2002) Honey: A potent gent for woun heling? J. Woun Ostomy Continene Nurs., 29(6): 295-300. 26. Skri, F.M. (2012) Mu n Khsitny, Suplemen Seht Tnp Efek Smping. 1 st e. Dinr Pustk Yogykrt, Inonesi. p11-15. Veterinry Worl, EISSN: 2231-0916 715