ABSTRACT
I N TROD UCTI ON
Bleeding can cause signiicant morbidity and m or t alit y in clinical set t ings. Several haem ost at ic agent s hav e been inv est igat ed for t heir r ole in haem ost asis6,9,11,13.
Ankaferd Blood St opper® ( ABS; Ankaferd Healt h Pr oduct s Lt d., I st anbul, Tur k ey ) is a t radit ional f ol k m ed i ci n al p l an t ex t r act p r od u ct t h at h as been appr ov ed in t h e m an agem en t of ex t er n al h em o r r h a g e a n d d en t a l su r g er y b l eed i n g s i n Tur key. ABS com pr ises a st andar dized m ixt ur e of
Effect s of folk m edicinal plant ext ract Ankafer d
Blood St opper
®
on ear ly bone healing
Sabri Cemil İşLER1, Sabit DEMIRCAN2, Sırmahan ÇAKARER1, Zerrin ÇEBI3, Cengizhan KESKIN3, Merva SOLUK4,
Emir YÜzBAşIOĞLU5
1- DDS, PhD Research Assistant, Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. 2- DDS, Research Assistant, Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. 3- DDS, PhD Professor, Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. 4- DDS, Research Assistant, Department of Tumor Pathology & Cytology, Institute of Oncology, Istanbul University, Istanbul, Turkey.
5- DDS, PhD, Private Practice, Istanbul, Turkey, Former Research Assistant, Department of Prosthodontics, Faculty of Dentistry, Ondokuz Mayıs University,
Samsun, Turkey.
Corresponding address: Dr. Sabit Demircan,DDS - İstanbul Üniversitesi Diş Hekimliği Fakültesi - Ağız, Diş, Çene Hastalıkları ve Cerrahisi Anabilim Dalı -
34093 Çapa-Fatih, Istanbul / Turkey - Phone: +90 212 414 20 20 – 30382 - Fax : +90 212 631 22 49 - e-mail: sabitdemircan@hotmail.com
Received: January 15, 2009 - Modiication: September 05, 2009 - Accepted: April 9, 2010
Obj ect iv e: Sev eral haem ost at ic agent s ar e available for clinical use. Ank afer d Blood St opper® (ABS), a mixture of ive medicinal plant extracts, has been used historically as
a haem ost at ic agent . The aim of t his in vivo st udy was t o invest igat e t he effect s of ABS on ear ly bone healing using a rat t ibia defect m odel. Mat er ial and Met hods: Sixt een m ale Wist ar rat s w er e random ized int o t w o gr oups of 8 anim als each. Aft er deep anest hesia w it h ket am ine, bone defect s ( 3 m m diam et er and 2 m m deep) w er e cr eat ed in t he r ight and left t ibiae of all anim als and eit her t r eat ed w it h 1 cc of ABS ( Gr oup 1) or left unt r eat ed
(Group 2; control). Surgical areas were closed primarily. The animals were sacriiced on
t he 7t h post operat ive day and bone sam ples w er e collect ed fr om t he t ibias. The sam ples
were examined histopathologically for infection, necrosis, ibrosis, new bone formation and
for eign body r eact ion. The hist om or phom et r ic r esult s w er e analyzed st at ist ically by t he chi
square test, with the level of signiicance set at p<0.05. Results:Signiicant differences were found in both groups in terms of inlammation, necrosis and new bone formation
( p= 0.001, p= 0.0001, p= 0.001) . No foreign body react ion was observed in t he experim ent al
group. ABS application decreased ibrosis in the experimental group, but there were no statistically signiicant differences from the control group. Conclusions: Histopathologically, it was observed that the application of ABS decreased the occurrence of inlammation and
necr osis, w hile incr easing new bone for m at ion in ear ly bone healing per iod. Fur t her in vit r o and in vivo studies are necessary for evaluating the beneits and possible adverse effects of t he applicat ion of t his her bal pr oduct on w ound healing.
Ke y w or ds: Ankafer d Blood St opper® ( ABS) . Thym us vulgar is. Glycyr r hiza glabr a. Vit is
vinifera. Alpinia oficinarum. Urtica dioica. Her bal m edicines. Bone healing.
t he plant s Thy m us v ulgar is, Gly cy r r hiza glabr a, Vitis vinifera, Alpinia oficinarum and Urtica dioica. Sev eral st udies hav e show n t hat each of t hese plant s has som e effect on t he endot helium , blood cells, angiogenesis, cellular pr oliferat ion, vascular dynam ics and cell m ediat ors2,3,7,8,10,12.Göoker, et al.5 ( 2008) invest igat ed t he haem ost at ic effect s of ABS and r epor t ed it s t herapeut ic pot ent ial t o be used for t he m anagem ent of haem or r hage5.
in clinical dent ist r y6,13, t her e is no evidence about t he effect s of ABS in vivo ex per im ent al m odels. The aim of t his in vivo st udy was t o invest igat e t he effect s of Ankafer d Blood St opper® on ear ly bone healing using a rat t ibia defect m odel.
M ATERI AL AN D M ETH OD S
An im a ls a n d Su r ge r y
Th e st u d y w as car r i ed ou t i n t h e I st an b u l Un i v e r si t y, Fa cu l t y o f D e n t i st r y, D e p a r t m e n t of Oral & Max illofacial Sur ger y and I nst it ut e of On co l o g y, D ep ar t m en t o f Tu m o r Pat h o l o g y & Cyt ology. Tr eat m ent of t he exper im ent al anim als was appr oved by t he I st anbul Univer sit y Anim al Resear ch an d Et h ics Com m it t ee. Ex p er im en t al an im als w er e obt ain ed fr om Th e Labor at or y of Exper im ent al Anim als, DETAM, I st anbul, Tur key.
A t ot al of 16 t w ent y- w eek- old m ale Wist ar rat s w eighing 250 t o 300 g w er e used in t his st udy and random ly assigned t o t wo groups of 8 anim als each. Prior t o surgery, t he anim als were anest het ized wit h a 0.7 m L int ram uscular ly inj ect ion of a solut ion cont aining xylazine hydrochloride ( Rom pun®, Bayer, Leverkusen, Germ any) and ket am ine hydrochloride ( Ket alar ; Pf izer, New Yor k , NY USA) at 1 / 0 . 5 pr opor t ion, 0.1 m L/ 100 g body w eight . Sur ger y was per for m ed under st er ile condit ions.
I n t he m id t ibia of rat s, a 5- m m long st raight longit udinal skin incision was done on t he front skin and, aft er m uscle split t ing ( plane- by- plane m uscle dissect ion) , t he per iost eal m em brane was st r ipped aw ay t o ex p ose b on e su r f ace. A st an d ar d ized ellipsoid r ound bone defect ( 5 m m in lengt h, 1 m m in height , 1 m m in dept h) was cr eat ed at t he ant erior port ion of t he diaphysis of bilat eral t ibias, 6 m m below t he knee j oint using a r ound car bide bur ( SS Whit e, Lakewood, NJ, USA) . The defect size was conirmed by a surgical stainless steel stent with t he cor r esponding dim ensions. The sur gical st ent was placed in the defect and conirmed visually by checking congr uit y t o t he defect wall.
Gr o u p 1 r e ce i v e d 1 cc o f An k a f e r d Bl o o d St opper® at t he t im e of sur ger y, w hile Gr oup 2 r eceived no t r eat m ent and ser ved as t he cont r ol. The muscles were sutured with 4/0 catgut (Doğsan, Istanbul, Turkey), and the laps were carefully reposit ioned and sut ured wit h 3/ 0 black silk sut ures (Doğsan, Istanbul, Turkey). Antibiotic (Sefazol, Must afa Nevzat , Tur key) was given t o t he anim als as an int ram uscular inj ect ion int raoperat ively and dur ing 3 day s post operat ively. No post operat ive com plicat ions w er e not iced dur ing t he post sur gical cour se. All anim als sur vived t hr oughout t he st udy per iod.
Th e r a t s o f ea ch g r o u p w er e h o u sed i n t o separat e cages w it h t w o or t hr ee anim als under clim at e- cont rolled condit ions ( 12 h light / 12 h dark;
t h er m ost at ically r eg u lat ed r oom t em p er at u r e) w it hout any rest rict ion of m obilizat ion. The anim als of each group were sacriiced with an overdose of ket am ine hydr ochlor ide ( 50 m g/ kg) on t he 7t h day aft er sur ger y, and t he defect s t oget her w it h sur r ounding bone w er e im m ediat ely r em oved for hist opat hological analysis.
Tissu e Pr e p a r a t ion a n d H ist op a t h olog ica l Ex a m in a t ion
Th e sp eci m en s w er e f i x ed i n 1 0 % n eu t r al b u f f er ed f or m alin ov er n ig h t at 4 °C, r in sed in phosphate buffered saline and decalciied in 20% for m ic acid solut ion ( Mer ck, Dar m st adt , Ger m any) f o r 1 0 d ay s. Th e d ecal ci f i ed sp eci m en s w er e embedded in parafin and cut into 20 semi-serial sect ions using a m icr ot om e ( Leica Micr osyst em ic, Ger m an y ) , an d r ou t in e h em at ox y lin an d eosin ( HE) st aining and Mallor y Tr ichr om e st aining w er e per for m ed. The sect ions w er e exam ined w it h light microscope under 40, 100 and 200x magniication ( Nikon Eclipse E600, Japan) . A hist om or phological r ev iew w as per for m ed by a sin gle blin ded or al pat hologist t o evaluat e t he pr esence of infect ion, necrosis, ibrosis, new bone formation, and foreign body r eact ion. The scor es for infect ion, necr osis, f ib r osis an d n ew b on e f or m at ion scor es w er e det er m ined by count ing t he associat ed cells and t heir rat io t o t he t ot al cell count in a st andar dized area at 40x magniication. The ratio of cells between 0- 25% was scor ed as none, 25- 50% as slight , 50-75% as m oder at e, and 75- 100% as advanced.
St a t ist ica l a n a ly sis
The st at ist ical differ ences bet w een t he cont r ol and t est gr oups w er e com par ed by chi squar e t est using t he GraphPad Prism aV.3 ( GraphPad Soft ware, Inc., USA) and the critical level of signiicance was P < 0.05.
RESULTS
The scores and percentages of inlammation, necrosis, ibrosis, and new bone formation in both gr oups ar e pr esent ed in Table 1 and illust rat ed in Figur es 1 and 2. Com par isons bet w een t he t est and control groups indicate a signiicant variability in the scores of inlammation, necrosis and new bone for m at ion ( p< 0. 001, p< 0. 0 001, p< 0. 001, r espect ively) . No for eign body r eact ions w er e seen in eit her of t he gr oups.
There was statistically signiicant difference bet w een t h e gr ou ps as for t h e n ecr osis scor es ( p < 0 . 0 0 0 1 ) . I n t h e t est g r ou p, 9 0 . 9 % of t h e specim en s did n ot sh ow n ecr osis, w h ile in t h e con t r ol g r ou p slig h t , m od er at e an d ad v an ced
necr osis was obser ved in 6.3% , 87.5% and 6.3% of t he specim ens, r espect ively.
Both groups showed similar range of ibrosis scores with no statistically signiicant difference ( p= 0.171) bet w een t hem .
Table 1- Inlammation, necrosis, ibrosis and bone formation scores in the test and control groups
Inlammation % n % n
None 63.6 7 0.0 0
Slight 36.4 4 62.5 10
Moderate 0.0 0 37.5 6
Advanced 0.0 0 0.0 0
χ²:15.16 p=0.001
Necrosis % n % n
None 90.9 10 0.0 0
Slight 9.1 1 6.3 1
Moderate 0.0 0 87.5 14
Advanced 0.0 0 6.3 1
χ²:24.92 p=0.0001
Fibrosis % n % n
None 9.1 1 0.0 0
Slight 27.3 3 62.5 10
Moderate 54.5 6 37.5 6
Advanced 9.1 1 0.0 0
χ²:5.01 p=0.171
Bone formation % n % n
None 0.0 0 31.3 5
Slight 18.2 2 68.8 11
Moderate 45.5 5 0.0 0
Advanced 36.4 4 0.0 0
χ²:19.99 p=0.0001
Control group Test group
Control group Test group
Control group Test group
Control group Test group
Figure 1- Comparison of inlammation, necrosis, ibrosis and bone formation scores in test group in percentage
The r esult s show ed t hat bone for m at ion scor es were signiicantly higher in the test group than in t he cont rol group ( p< 0.0001) . I n t he group t reat ed w it h ABS, slight , m oderat e and advanced bone f or m at ion w as obser v ed in 1 8 . 2 % , 4 5 . 5 % an d 36.4% of t he specim ens, r espect ively.
Repr esent at ive hist ological sect ions of var ious
specim ens in t he t est group t hat show ed decreased inlammation and necrosis, and increased new bone form at ion in early bone healing period are illust rat ed in Figur es 3 and 4.
Figure 2- Comparison of inlammation, necrosis, ibrosis and bone formation scores in control group in percentage
Figure 3- (a) Non-remodeled newly formed bone tissue covering the defect area and illing the medullar space
D I SCUSSI ON
Bl e e d i n g ca n ca u se si g n i f i ca n t m o r b i d i t y an d m or t alit y in an y clin ical set t in g . Bleed in g m anagem ent has been st udied ex t ensiv ely and various haem ost at ic agent s are available for clinical use6,13.
ABS i s a f o l k l o r i c m e d i ci n a l p l a n t e x t r a ct p r o d u ct , w h i ch h a s h i st o r i ca l l y b een u sed i n Tu r k ish t r ad it ion al m ed icin e as a h aem ost at ic agent . I t is a st andar dized m ixt ur e of t he plant s
T. vulgar is, G. glabr a, V. vinifer a, A. oficinarum
an d U. dioica, each of w h ich h as som e ef f ect on hem at ological and vascular param et er s, and cel l u l a r p r o l i f er a t i o n2 , 3 , 7 , 8 , 1 0 , 1 2. Ea ch i n g r ed i en t of this mixture has speciic characteristics. G. glabr a inhibit s angiogenesis, decr eases vascular
endot helial gr ow t h fact or pr oduct ion and cyt okine-in du ced n eov ascu lar izat ion . G. glabr a also h as an t i i n f l am m at o r y, an t i - t h r o m b i n , an t i p l at el et , an t iox idan t , an t i- at h er oscler ot ic, an d ant it u m or act ivit ies10. T. vulgar is has been show n t o exhibit v a r y i n g l ev el s o f a n t i - ox i d a n t a ct i v i t y, w h i ch m ay help t o pr ev ent in v iv o ox idat iv e dam age,
s u c h a s l i p i d p e r o x i d a t i o n , a s s o c i a t e d w i t h a t h e r o scl e r o si s7. I n o cu l a t i o n e x p e r i m e n t s o n det ached leaves of V. vinifer a exhibit ed enhanced r esist ance t ow ar ds pat hogens2, 3.V. v inifer a also has ant i- at her oscler ot ic and ant it um or effect s14,15. A. oficinarum inhibit s nit r ic ox ide pr oduct ion in lip op oly sacch ar id e act iv at ed m ou se p er it on eal m acr ophages8. U. dioica can pr oduce hypot ensive responses t hrough a vasorelaxat ion effect m ediat ed by t he r elease of endot helial nit r ic oxide and t he open in g of pot assiu m ch an n els, an d t h r ou gh a negat ive inot r opic act ion12.
Gok er, et al.5 ( 2 0 0 8 ) sh ow ed t h at t h e ABS-i n d u ce d n e t w o r k f o r m a t ABS-i o n ABS-i s r e l a t e d t o t h e funct ions of blood pr ot eins and r ed blood cells. The basic m echanism of act ion for ABS appear s t o be t he for m at ion of an encapsulat ed pr ot ein net w or k t hat pr ovides focal point s for er yt hr ocyt e a g g r e g a t i o n . B l o o d c e l l s ( e r y t h r o c y t e s a n d plat elet s) also aggr egat ed and par t icipat ed in t he net w or k for m at ion, w it h t he er yt hr ocyt es for m ing a m ass. Exposur e t o ABS seem s t o pr ovide a t issue oxygenat ion as w ell as a physiological haem ost at ic pr ocess w it hout affect ing any indiv idual clot t ing
Figure 4- (a) New bone tissue layer separated from the defect wall by apposition lines, which is located in the loose connective
tissue with mild inlammatory cell iniltration at the apex of the defect fragment (Hematoxylin-Eosin (HE) x200). (b) Areas of endochondral ossiication in an active connective tissue at the defect area (HE x200). (c) Subperiostal ossiication and osteoprogenitor cells that are proliferated and transforming into osteoblasts underneath the periosteum, which is traumatized near the defect area (HE x200). (d) Vessel-rich active connective tissue formed by fusiform cells underneath the surface epithelium (HE x200).
fact or. This unique m echanism of act ion pr ovides ABS w it h an advant age over ot her haem ost at ically act ive plant ext ract s1,4.
Th e h ist opat h ological r esu lt s of t h e pr esen t st udy show ed t hat over sixt y per cent of t he defect s treated with ABS were free of inlammation, which is probably related to the antiinlammatory activity of som e com ponent s of t he haem ost at ic agent . Although the occurrence of ibrosis was statistically sim ilar in bot h gr ou ps, t h e ABS- t r eat ed gr ou p showed lower ibrosis rate than the non-treated con t r ol gr ou p, w h ich m ay be at t r ibu t ed t o t h e incr eased speed of healing in t he t est gr oup.
The defect s t r eat ed w it h ABS also show ed m or e int ense new bone for m at ion and less occur r ence of necr osis, w hich m ay be r elat ed t o t he incr eased sp eed of h ealin g an d d ecr eased in f lam m at ion w hich is associat ed w it h ant ioxidant act ivit y of t he com ponent s of t he ABS.
CON CLUSI ON
Wit hin t he lim it at ions of t his st udy, t he follow ing co n cl u si o n s w e r e d r a w n : 1 . ABS d e cr e a se d the inlammation and necrosis process; 2. ABS incr eased t he new bone for m at ion in ear ly bone healing per iod; 3. No for eign body r eact ion t o ABS was observed; 4. Furt her in vit ro and in vivo st udies ar e n ecessar y t o assess b en ef it s an d p ossib le adverse effect s of t he applicat ion of Ankaferd Blood St opper® on w ound healing.
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