A Comparison of the Effects of Grayanotoxin- Containing Honey (Mad Honey), Normal Honey, and Propolis on Fracture Healing

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1 Originl Pper Receive: July 4, 2017 Accepte: Februry 11, 2018 Publishe online: Februry 11, 2018 A Comprison of the Effects of Grynotoxin- Contining Honey (M Honey), Norml Honey, n Propolis on Frcture Heling Aynur Shin Suh Turkmen b Nizmettin Guzel c Ahmet Mentese Suleymn Turei Suleymn Cner Krhn Esin Yulug e Selim Demir f Osmn Aynci c Orhn Deger Abulkir Gunuz Deprtment of Emergency Meicine, Fculty of Meicine, Kreniz Technicl University, Trbzon, Turkey; b Deprtment of Emergency Meicine, Fculty of Meicine, Acıbem University, Istnbul, Turkey; c Deprtment of Orthopeics n Trumtology, Smsun Trining n Reserch Hospitl, Smsun, Turkey; Deprtment of Biochemistry, Fculty of Meicine, Kreniz Technicl University, Fculty of Meicine, Trbzon, Turkey; e Deprtment of Histology n Embryology, Fculty of Meicine, Kreniz Technicl University, Trbzon, Turkey; f Deprtment of Nutrition n Dietetics, Fculty of Helth Sciences, Kreniz Technicl University, Trbzon, Turkey Significnce of the Stuy Although severl methos re vilble for ccelerting the heling process, frcture heling is growing helth problem. This is the first experimentl stuy tht revels the time-epenent beneficil effects of grynotoxin n propolis on frcture heling s ssesse by riogrphy n histology. Keywors Grynotoxin Propolis Honey Frcture heling Abstrct Objectives: Delye heling n non-union of frctures hve significnt effect upon ptient morbiity. Stuies hve therefore lrgely concentrte on ccelerting frcture heling. This stuy ws intene to compre the effect of m honey n propolis on frcture heling using riologicl n histopthologicl nlysis. Subjects n Methos: Femur frcture ws surgiclly performe on 48 rts, followe by fixtion. Animls were then ivie into 8 groups: 2 control groups (15- n 30-y) n 6 tretment groups (15- n 30-y norml honey, 15- n 30-y m honey, n 15- n 30-y propolis). Rts were scrifice t the en of these perios, n riologicl n histologicl exmintions were performe. Results: Riologicl heling in the propolis group fter 15-y therpy ws sttisticlly better thn in the control (p = 0.004) n norml honey (p = 0.006) groups. After 30-y therpy, heling in the propolis group (p = 0.005) n grynotoxin-contining m honey group (p = 0.007) were significntly better thn in the control group. Histologiclly, there ws sttisticlly significnt ifference between the 15-y propolis group n the other groups (control, honey, m honey: p = 0.003, p = 0.003, n p = 0.002, respectively). We lso foun sttisticlly significnt ifference when the 30-y propolis group (p = 0.005) n m honey group (p = 0.007) were compre to the control group. Conclusions: This stuy shows tht grynotoxin-contining m honey n propolis cn ccelerte frcture heling The Author(s) Publishe by S. Krger AG, Bsel E-Mil krger@krger.com The Author(s) Publishe by S. Krger AG, Bsel This is n Open Access rticle license uner the Cretive Commons Attribution-NonCommercil-4.0 Interntionl License (CC BY-NC) ( pplicble to the online version of the rticle only. Usge n istribution for commercil purposes requires written permission. Aynur Shin Deprtment of Emergency Meicine, Fculty of Meicine Kreniz Technicl University TR Trbzon (Turkey) E-Mil hotmil.com

2 Introuction Mterils n Methos Stuy Design This rnomize, controlle, nonbline experimentl stuy ws performe fter Institutionl Animl Cre n Ethics Committee (2011/23) pprovl ws obtine. The stuy ws performe in ccornce with guielines for the housing n cre of lbortory nimls. Stuy Setting n Popultion Forty-eight femle Sprgue Dwley rts weighing g were use. The nimls were house uner stnr light/rk conitions (12-h light n 12-h rk) t constnt temperture (22 C) n humiity (55 + 5%) with unrestricte ccess to stnr lbortory niml chow n wter. Stuy Protocol Rts were rnomly ssigne to 8 groups of 6 members ech 2 control groups (15-y control, 30-y control) n 6 tretment groups (15-y propolis, 30-y propolis, 15-y norml honey, 30-y norml honey, 15-y m honey, n 30-y m honey). The sme surgicl proceure ws performe on ll groups before tretment. The rts in the tretment groups were trete ily. Surgicl Proceure Imges of the surgicl proceure re shown in Figure 1. Rts were fste for 4 h before surgery. For inuction of nesthesi, 5 mg/kg xylzine hyrochlorie (Rompun ; Byer Helthcre, Color version vilble online Setbcks in frcture heling re growing helth problem. These hve n verse psychologicl impct n lso result in workforce n economic losses. Vrious methos of ccelerting frcture heling n permitting return to ily life s erly s possible re currently uner investigtion [1]. Grynotoxin (GTX) is foun in the flower prts of plnt species such s Rhooenron n Klmi. GTX is present in rhooenron pollens. It is therefore lso foun in honeys prouce from this regionl plnt. This toxin-contining honey is known loclly s m honey [2]. People in this region use loclly prouce honey s n lterntive therpy for woun heling, to tret rthritis, stomch pin, intestinl iseses, n hypertension, n to increse sexul performnce [3, 4]. Propolis is complex, hesive prouct me by bees from vrious plnt sees, leves or stems n plnt secretions [1]. The min compouns it contins re flvonois, which hve ntioxint chrcteristics [5]. Propolis hs ttrcte consierble interest in recent yers ue to its phrmcologicl n biologicl effects. Stuies hve shown tht it possesses nticncer, heptoprotective, nti-inflmmtory, n ntioxint ctivities [6, 7]. Only 1 experimentl stuy hs emonstrte positive effects of propolis in frcture heling [8]. However, the effects of GTX in loclly prouce m honey, use s n lterntive therpy in woun heling, hve not been investigte in frcture heling. The purpose of this stuy ws to investigte n compre the effects of GTX-contining m honey n propolis on frcture heling, using riology n histology, in n experimentl femur frcture moel. Fig. 1. Surgicl proceure. 100 b c Shin et l.

3 Leverkusen, Germny) n 50 mg/kg ketmine hyrochlorie (Ketlr ; Pfizer, Istnbul, Turkey) were ministere by the intrperitonel route. Following pproprite clening n covering of the surgicl fiel, the skin of the right thigh ws opene pproximtely 2 cm from the lterl spect with longituinl incision (Fig. 1). The femur ws ccesse. A trnsverse frcture ws then me using bttery-powere rill with 0.8 mm Kirschner wire (Hipokrt, Izmir, Turkey) (Fig. 1b). The wire ws retrcte istlly, n once the frcture line h been reuce it ws move in proximl irection in retrogre mnner. The wire ws gin move forwr with the tip of nother wire so tht it woul be completely burie (Fig. 1c). Once fixtion h been estblishe, the ccurcy of the frcture line reuction ws confirme using X- ry imging (Fig. 1). On ys 15 n 30, s pplicble, rts in the control n tretment groups were scrifice by cervicl isloction uner nesthesi. Right femurs were extrcte by isrticultion from the hip n knee. Riologicl imges were tken, n soft tissue specimens were store in 10% formlehye for preprtion for histologicl exmintion. Tretment Protocols Rts receive no ntibiotic therpy before or fter the surgicl proceure. No gent ws ministere for therpeutic purposes in the 15- or 30-y control groups. Ech rt in the propolis groups (15- n 30-y) receive 200 mg/kg propolis per y by the orogstric route throughout the stuy perio [8]. The propolis smples use in this stuy were prouce by honeybees (Apis mellifer L.) in Trbzon, Turkey, n were provie by the Trbzon Agriculturl Development Coopertive. For stock wter (50,000 mg/ml) preprtion, 1 g of propolis ws issolve in 20 ml wter n then incubte t 60 C n 150 rpm in n orbitl shker for 24 h. After incubtion, smples were centrifuge t 4000 rpm for 10 min. Superntnts were filtere through filter pper n 0.22 mm filters [7]. As the men mount of m honey consume for therpeutic purposes by 70-kg ult is 4 6 g/y (1 tespoonful), ech rt in the m honey groups (15- n 30-y) receive men 80 mg/kg m honey per y by the orogstric route throughout the stuy perio [1]. ws obtine from smple tht cuse clinicl cse of toxicity fter consumption; it ws confirme to hve originte from Rhooenron-contining re of the Blck Se Region. The mount of GTX it contine ws not mesure. Ech rt in the norml honey groups (15- n 30-y) receive 80 mg/kg norml honey per y by the orogstric route. The norml honey (Blprmk Blossom Honey, Izmir, Turkey) use in the experiments ws obtine from supermrkets n then subjecte to stnr tests in the prouction fcilities. Riologicl Evlution Riogrphic imges were score ccoring to the Lne-Snhu riologicl scoring system (Tble 1) [9]. Scoring ws performe by 2 inepenent orthopeists bline to the stuy groups. Tble 1. The Lne-Snhu riogrphic scoring system Degree of bone formtion No new bone forme 0 The re of new bone ccounts for 25% of the efect re 1 The re of new bone ccounts for 50% of the efect re 2 The re of new bone ccounts for 75% of the efect re 3 The re of new bone ccounts for 100% of the efect re 4 Degree of union Frcture line is fully visible 0 Frcture line is prtilly visible 2 Frcture line is not visible 4 Degree of meullry cvity remoeling No sign of remoeling 0 Recnliztion of the meullry cvity 2 Recnliztion of corticl bone 4 Tble 2. The Huo histologicl scoring system Frcture region finings Fibrous tissue only 1 Preominntly fibrous tissue 2 Equl mounts of fibrous n crtilge tissue 3 Preominntly crtilge tissue with little fibrous tissue 4 Crtilge tissue only 5 Preominntly crtilge tissue with little immture bone 6 Equl mounts of crtilge n immture bone tissue 7 Preominntly immture bone with little crtilge tissue 8 Heling with immture bone 9 Heling with mture bone 10 Histopthologicl Exmintion Tissue specimens were fixe in 10% neutrl formlin, renere trnsprent in xylene, n embee in prffin blocks. Sections 5 μm in thickness were me using fully utomtic microtome (Leic RM 2255, Tokyo, Jpn). Compouns were nlyze uner light microscopy (Olympus BX51, Jpn) by n experience histologist blin to the groups. Compouns from the groups were photogrphe using igitl cmer (Olympus DP71, Jpn) uner light microscope. The 10-point histologicl scoring system escribe by Huo n Troino [10] ws use for histologicl evlution of compouns (Tble 2). Sttisticl Anlysis Sttisticl nlysis ws performe using SPSS (Sttisticl Pckge for Socil Sciences for Winows v.13.0; SPSS, Chicgo, IL, USA) softwre. During evlution of riologicl n histologicl scores, the 15- n 30-y groups were seprtely compre mong themselves. Nonprmetric tests were use for t nlysis. The Kruskl-Wllis test ws use for multiple comprisons n the Mnn-Whitney U s post hoc test with Bonferroni correction. Vlues of p < fter correction were regre s significnt. Results Points Riologicl Scoring Fifteen- n thirty-y riologicl scoring results from the stuy groups re shown in Tbles 3 n 4. After Effects of M Honey n Propolis on Frcture Heling 101

4 Tble 3. Sttisticl nlysis between groups on y 15 Mein riologicl scores (95% CI) control norml honey propolis m honey 2.5 (2 6) 3.5 (2 7) 8.5 (7 11) 5 (4 8) Control * Norml honey 0.006* Propolis * Mnn-Whitney-U test, p vlues < (p vlue juste for Bonferroni correction) re regre s sttisticlly significnt. Tble 4. Sttisticl nlysis between groups on y 30 Mein riologicl scores (95% CI) control norml honey propolis m honey 9.5 (6 12) 12.5 (7 14) 14.5 (12 16) 13 (12 16) Control * 0.007* Norml honey Propolis * Mnn-Whitney-U test, p vlues < (p vlue juste for Bonferroni correction) re regre s sttisticlly significnt. 15 ys therpy, nimls receiving propolis exhibite significntly higher riologicl scores compre to the controls (p = 0.004) n subjects receiving norml honey (p = 0.006). There ws no significnt ifference between the 15-y m honey group n the control group (Tble 3). Group scores on the 15th y re shown in Figure 2. After therpy for 30 ys, the riologicl scores of nimls receiving propolis (p = 0.005) n m honey (p = 0.007) were significntly higher thn those of the control group (Tble 4). No sttisticlly significnt ifferences were observe between the riologicl scores of the norml honey group n the control group, or between the propolis n m honey tretment groups. Group scores on the 30th y re shown in Figure 3. Histopthologicl Comprison Evlution of histologicl gre in subjects receiving therpy for 15 ys revele no histologicl gre higher thn 3 in the control or norml honey groups, n the ifference between these two ws insignificnt. All subjects in the 15-y m honey group h histologicl scores of 4, while ll subjects in the 15-y propolis group h histologicl gre scores of 6 or bove (Fig. 4). A significnt increse in histologicl gre ws observe in the 15-y propolis group compre to the control (p = 0.003), 15-y norml honey (p = 0.003), n 15-y m honey (p = 0.002) groups. No significnt increse in histologicl gre ws observe in the 15-y m honey group compre to the 15-y norml honey or 15-y control groups. No histologicl score bove 8 ws observe in the control or norml honey groups t exmintion of histologicl gre fter 30 ys therpy. Histology scores of 8 or bove were observe in ll rts in the 30-y propolis n m honey groups. The highest histology gre, i.e., 10, ws etermine in 1 niml of the propolis group (Fig. 5). Figure 6 shows bone tissue smples from the tretment n control groups on ys 15 n 30. A significnt increse in histologicl gre ws observe in the 30-y propolis (p = 0.005) n 30-y m honey (p = 0.007) groups compre to the control group. Comprison of the 30-y propolis n 30-y norml honey groups revele sttisticlly significnt histologicl improvement in the propolis group (p = 0.008). There ws no significnt ifference between the 30-y m honey group n the 30-y propolis or 30-y norml honey groups. 102 Shin et l.

5 Riologicl score 8 6 Riologicl score Control Norml honey Propolis Groups Control Norml honey Propolis Groups Fig. 2. Specilist scores for the groups on the 15th y of the experiment. Fig. 3. Specilist scores of the groups on the 30th y of the experiment. 6 Histologicl gre Histologicl gre Number of cses Number of cses Control Honey Propolis Groups 0 Control Honey Propolis Groups Fig. 4. Histologicl gres on the 15th y of the experiment. Fig. 5. Histologicl gres on the 30th y of the experiment. Discussion This experimentl stuy revele positive effects of propolis n m honey on frcture heling in n experimentl setting, s shown by X-ry n histopthologicl stuies. The stuy finings my be interprete s inicting tht propolis is more effective thn m honey over both 15- n 30-y tretment perios. Evlution of the riologicl n histologicl scores of the propolis tretment group in our stuy showe significnt heling in both the 15- n 30-y experimentl nimls compre to the control group. Mny cellulr n biochemicl events such s neurohormonl n neurohumorl mechnisms re involve in frcture heling, n stuies hve shown tht free oxygen ricls hve eleterious impct on heling [11, 12]. Vrious rugs n Effects of M Honey n Propolis on Frcture Heling 103

6 Color version vilble online b c e f g h Fig. 6. Histologicl imges t the 15th y ( ) n 30th y (e h)., e Control. b, f Norml honey. c, g M honey., h Propolis. Crtilge tissue (c), fibrous tissue (f), immture bone tissue (b). Hemtoxylin eosin fctors hve been investigte for their bility to ccelerte frcture heling. One such is propolis, which possesses ntioxint properties. Propolis consists of 50% flvonoi- n phenolic ci-bse resin n vegetble blsm, 30% wx, 10% essentil oils, 5% pollen, n 5% other orgnic compouns. Flvonois in propolis re ntioxints tht ericte free oxygen ricls by onting hyrogen. In ition, flvonois complexe with iron ions suppress the Fenton rection tht cuses hyroxyl ricls [2]. Severl stuies hve emonstrte the ntioxint property of propolis in tissues. In histomorphometric stuy, Altn et l. [13] showe tht propolis stimulte new bone formtion in bone suture seprtion n increse inflmmtory cell numbers n cpillry numbers in tissues. Guney et l. [8] observe positive effect of propolis on frcture heling; they reporte tht while riologicl scores in the 3r n 6th weeks were better compre to the control group, histologicl scores were 104 comprble to those of the control group t the 6th week. Our propolis smples were collecte in Trbzon in the Northest of Turkey. Propolis is known to be rich in polyphenolic compouns, such s pinocembrin, islpinin, pinostropin, nringenin, quercetin, chrysin, pinobnksin, glngin, pigenin, 3,40,7-trimethoxy flvnone, n cffeic ci [14]. The positive effect of propolis escribe in the literture thus supports the finings of our stuy. When the efficcy of the m honey n control groups ws compre in terms of frcture heling, no significnt ifference ws observe s evience by riologicl n histopthologicl scores on y 15 of tretment. However, t the en of the 30-y protocol, both scoring results were significntly superior in the m honey group compre to the control group. As m honey is use to tret numerous iseses such s ibetes, hypertension, n hyperlipiemi, the mechnisms of ction of GTX in these pthologies hve been stuie [15 Shin et l.

7 17]. However, there re no reports on the effects of m honey on frcture heling. Severl mechnisms my unerlie the positive effects observe by us. First, bone metbolism events re ffecte by the nervous system. Meitors proucing this effect inclue glutmte, ctecholmines, leptin, n VIP [18]. Glutmte hs been shown to ply significnt role in bone-nerve interction in prticulr, being ssocite with other nerve mrkers in bone cells, n hs been reporte to ply n importnt role in remoeling fter mechnicl loing by blncing ctbolic n nbolic ctivities [19]. Kim et l. [20] reporte increse GABA n glutmte relese fter GTX ministrtion. Increse glutmte relese fter GTX intke my explin the positive effect on bone heling of m honey. Durgi et l. [21] showe tht GTX-3 inhibits cytosolic crbonic nhyrse 2 in the micromolr rnge in mmmls. Osteoclsts perform bone resorption through hyrogen relese by mens of crbonic nhyrse. A ecrese in the effect of crbonic nhyrse, n thus of osteoclsts, with the ministrtion of GTX my be possible cuse of the positive effect of m honey on frcture heling. This stuy showe tht GTX-contining m honey increse vsculrity in the frcture region. Yet nother reson for the effect of GTX on bone heling my be increse bloo flow in ssocition with peripherl vsoiltion. Sibel et l. [22] reporte tht GTX increse ntioxint ctivity in tissues in their experimentl stuy. The ntioxint property of GTX my therefore explin the positive effect on heling. Norml honey ws inclue in this stuy in orer to ssess its efficcy in terms of frcture heling of other chemicl compouns contine in propolis n m honey. Honey hs complex chemicl composition. It contins 25 crbohyrtes, free mino cis, vitmins, trce elements, flvonois, n ntioxint, ntimicrobil, n nti-inflmmtory substnces [23]. Aitionlly, substnces or toxins specific to the flower flor from which it is obtine my lso cuse iffering effects. Severl stuies hve investigte its use in woun heling n for treting coughs, for boosting the immune system, n for ntimicrobil ctivity [23, 24]. In our stuy, the effect on riologicl n histopthologicl scores in the norml honey group in both the 15- n 30-y protocols resulte in no significnt improvement in heling compre to the control group. Interestingly, no significnt ifference ws etermine between long-term riologicl score vlues in the propolis n m honey groups n those of the norml honey group. Limittions There re number of limittions to this stuy. The mount of GTX contine in the m honey use in the experimentl protocol ws not etermine using quntittive metho. One of the min limittions of this stuy is the bsence of ny investigtion of ose response. Bloo prmeters showing the ntioxint chrcteristic of GTX n fctors incresing bloo supply were lso not investigte. Biomechnicl testing using the contrlterl, nonfrcture femur s n internl control ws lso not performe. Conclusions This experimentl stuy revels time-epenent beneficil effect of GTX n propolis on frcture heling s evlute by riogrphic n histologicl ssessment. It lso shows tht GTX n propolis hve similr riologicl n histologicl therpeutic effects in bone frcture over perio of 30 ys. Further stuies re neee to clrify the effects of GTX on frcture heling. References 1 Victori G, Petrisor B, Drew B, et l: Bone stimultion for frcture heling: Wht s ll the fuss? Inin J Orthop 2009; 43: Gunuz A, Turei S, Russell RM, et l: Clinicl review of grynotoxin/m honey poisoning pst n present. Clin Toxicol 2008; 46: Ttli O, Krc Y, Turkmen S, et l: The effect of m honey on testosterone levels of mle rts. Brtisl Lek Listy 2016; 117: Gunuz A, Turei S, Uzun H, et l: poisoning. Am J Emerg Me 2006; 24: Co XP, Chen YF, Zhng JL, et l: Mechnisms unerlying the woun heling potentil of propolis bse on its in vitro ntioxint ctivity. Phytomeicine 2017; 34: Wgh VD: Propolis: A woner bees prouct n its phrmcologicl potentils. Av Phrmcol Sci 2013; 2013: Demir S, Aliyzicioglu Y, Turn I, et l: Antiprolifertive n propoptotic ctivity of Turkish propolis on humn lung cncer cell line. Nutr Cncer 2016; 68: Guney A, Krmn I, Oner M, et l: Effects of propolis on frcture heling: n experimentl stuy. Phytother Res 2011; 25: Lne JM, Snhu HS: Current pproches to experimentl bone grfting. Orthop Clin North Am 1987; 18: Huo MH, Troino NW: The influence of ibuprofen on frcture repir: biomechnicl, biochemicl, histologic, n histomorphometric prmeters in rts. J Orthop Res 1991; 9: Effects of M Honey n Propolis on Frcture Heling 105

8 11 Khllf FG, Kehine EO, Hussein S: Bone heling n hormonl biossy in ptients with long-bone frctures n concomitnt he injury. Me Princ Prct 2016; 25: Shui AN, Mohm S, Muhmm N, et l: Effects of α-tocopherol on the erly phse of osteoporotic frcture heling. J Orthop Res 2011; 29: Altn BA, Kr IM, Nlcci R, et l: Systemic propolis stimultes new bone formtion t the expne suture: histomorphometric stuy. Angle Ortho 2013; 83: Uzel A, Sorkun K, Oncg O, et l: Chemicl compositions n ntimicrobil ctivities of four ifferent Antolin propolis smples. Microbiol Res 2005; 160: Gunuz A, Klkn A, Turei S, et l: Pseuocholinesterse levels re not ecrese in grynotoxin (m honey) poisoning in most ptients. J Emerg Me 2012; 43: Gunuz A, Eryın I, Turkmen S, et l: Anlgesic effects of m honey (grynotoxin) in mice moels of cute pin n pinful ibetic neuropthy. Hum Exp Toxicol 2014; 33: Oztsn N, Altınkynk K, Akcy F, et l: Effects of m honey on bloo glucose n lipi levels in rts with streptozocin-inuce ibetes. Turk J Vet Anim Sci 2005; 29: Jones KB, Mollno AV, Morcuene JA, et l: Bone n brin: review of neurl, hormonl n musculoskeletl connections. Iow Orthop J 2004; 24: Skerry TM, Tylor AF: Glutmte signlling in bone. Curr Phrm Des 2001; 7: Kim SE, Shin MC, Akike N, et l: Presynptic effects of grynotoxin on excitory n inhibitory nerve terminls in rt ventromeil hypothlmic neurons. Neurotoxicology 2010; 31: Durgi S, Scozzfv G, Vullo D, et l: Inhibition of mmmlin crbonic nhyrses I- XIV with grynotoxin III: solution n in silico stuies. J Enzyme Inhib Me Chem 2014; 29: Sibel S, Enis YM, Hüseyin S, et l: Anlysis of gryntoxin in Rhooenron honey n effect on ntioxint prmeters in rts. J Ethnophrmcol 2014; 156: Ouwole O, Meremikwu MM, Oyo-It A, et l: Honey for cute cough in chilren. Cochrne Dtbse Syst Rev 2012:CD Zbuche A: Up-to-te use of honey for burns tretment. Ann Burns Fire Dissters 2014; 27: Shin et l.