Total Polyphenol Content and Minimum Inhibitory Concentration of Pomegranate ( Punica granatum Linn ) Extracts Against Oral Microorganisms

Diet plays an important role in preventing oral diseases including dental caries, dental erosion, developmental defects, oral mucosal diseases and, to lesser extent, peri­ odontal diseases [1]. Diet also influences the development of oral cavity. Depending on whether there is an early or late nutritional imbalance, consequences are certainly dif­ ferent. In fact, an early nutritional imbalance influences malformations [2]. Vitamin and mineral deficiency in the phase before conception influences the development of future embryo, dental organogenesis, the growth of max­ illa, as well as skull/facial development [3]. Therefore, dietary intervention with functional foods is required to maintain oral health and alleviate oral diseases. Oral cavity is inhabited by more than 700 microbial species and many intrinsic and extrinsic factors affect the composition, metabolic activity and pathogenicity of oral microflora [4, 5]. It is well established that many metabolites produced by plant extracts such as tannins, terpenoids, alkaloids, and flavonoids provide new source of antimicrobial substances that help in combating new developing drug resistant pathogens [6]. There are three main reasons to be interested in the healing power of plant extracts: first, pharmacological studies have demonstrat­ ed that many plants are known to possess antimicrobial agents; second, there are many side effects associated with over prescription of antibiotics; and third, the number of antibiotic resistant microorganisms is increasing [7]. Polyphenols represent the largest group of phyto­ chemicals found in plants, particularly in fruits, seeds, and leaves. These compounds exhibit health benefits through complementing and adding to the function of antioxidant vitamins and enzymes as defense against oxidative stress caused by excess reactive oxygen species. Dietary polyphe­ nols have been paid attention due to their benefits on hu­ man health. Frequent intake of fruits rich in polyphenols has been linked to lowered risks of many diseases [8]. Health benefit of pomegranate extracts in reducing pathogenic oral bacteria and risk of plaque formation, gingivitis, and periodontal disease has been shown in hu­ man clinical trials [9]. Primary bioactive constituents of pomegranate juice are polyphenols that have potent anti­ oxidant characteristics [10]. Punica granatum, commonly known as pomegranate, is a member of the Punicaceae (Pomegranate family) [11]. The pomegranate tree typi­ cally grows 12­16 feet and has many spiny branches. The ripe pomegranate fruit can be up to five inches wide with a deep red, leathery skin, grenade­shaped, and crowned by the pointed calyx. The fruit contains many seeds (Arils) separated by white, membranous pericarp, and each is surrounded by small amounts of tart, red juice. Leaves are shiny about 7.6 cm long [12]. Punicalagin is a major antioxidant polyphenol in pomegranate juice [13]. Total Polyphenol Content and Minimum Inhibitory Concentration of Pomegranate (Punica granatum Linn) Extracts Against Oral Microorganisms


INTRODUCTION
Diet plays an important role in preventing oral diseases including dental caries, dental erosion, developmental defects, oral mucosal diseases and, to lesser extent, peri odontal diseases [1].Diet also influences the development of oral cavity.Depending on whether there is an early or late nutritional imbalance, consequences are certainly dif ferent.In fact, an early nutritional imbalance influences malformations [2].Vitamin and mineral deficiency in the phase before conception influences the development of future embryo, dental organogenesis, the growth of max illa, as well as skull/facial development [3].Therefore, dietary intervention with functional foods is required to maintain oral health and alleviate oral diseases.
Oral cavity is inhabited by more than 700 microbial species and many intrinsic and extrinsic factors affect the composition, metabolic activity and pathogenicity of oral microflora [4,5].It is well established that many metabolites produced by plant extracts such as tannins, terpenoids, alkaloids, and flavonoids provide new source of antimicrobial substances that help in combating new developing drug resistant pathogens [6].There are three main reasons to be interested in the healing power of plant extracts: first, pharmacological studies have demonstrat ed that many plants are known to possess antimicrobial agents; second, there are many side effects associated with over prescription of antibiotics; and third, the number of antibiotic resistant microorganisms is increasing [7].
Polyphenols represent the largest group of phyto chemicals found in plants, particularly in fruits, seeds, and leaves.These compounds exhibit health benefits through complementing and adding to the function of antioxidant vitamins and enzymes as defense against oxidative stress caused by excess reactive oxygen species.Dietary polyphe nols have been paid attention due to their benefits on hu man health.Frequent intake of fruits rich in polyphenols has been linked to lowered risks of many diseases [8].
Health benefit of pomegranate extracts in reducing pathogenic oral bacteria and risk of plaque formation, gingivitis, and periodontal disease has been shown in hu man clinical trials [9].Primary bioactive constituents of pomegranate juice are polyphenols that have potent anti oxidant characteristics [10].Punica granatum, commonly known as pomegranate, is a member of the Punicaceae (Pomegranate family) [11].The pomegranate tree typi cally grows 1216 feet and has many spiny branches.The ripe pomegranate fruit can be up to five inches wide with a deep red, leathery skin, grenadeshaped, and crowned by the pointed calyx.The fruit contains many seeds (Arils) separated by white, membranous pericarp, and each is surrounded by small amounts of tart, red juice.Leaves are shiny about 7.6 cm long [12].Punicalagin is a major antioxidant polyphenol in pomegranate juice [13].
This study aimed to analyze the total polyphenol con tent of different parts of pomegranate extracts: seed, rind, pith and crude.The study also assessed the minimum in hibitory concentration (MIC) of different pomegranate parts against oral microorganisms.

Collection of fruits
The Kabul pomegranate was purchased from the local market in Chennai, India.It was cut by making a shallow slit on the top of the pomegranate where the knob/stem is (this part is known as crown).The shallow circle was created by cutting all the way around the rind.The inner seeds were revealed by pulling the crown of pomegran ate.Three shallow slits were made by cutting the outer rind, followed by three of the white pith lines, from the top to the bottom of the fruit.Three large sections were created by pulling the fruit.These sections were merged in a large bowl of cold water.Seeds were separated from rind and pith and drained in a colander to remove any additional pith.

Preparation of methanolic extracts
Methanolic rind extract (MRE) was prepared by cutting rind into small squares (approximately 5 mm 2 ) that were dried at 55°C for 24 hours, and stored in an air tight con tainer in dark until further use, 10 g each of the dried material was taken in a sample.It is then extracted with methanol using Soxhlet extractor for 8 hours.Extract is then after distilled to remove solvent and used for testing the antimicrobial activity.
Methanolic seed extract (MSE) was prepared by dry ing seeds at 55°C for 24 hours and then after following the same procedure as for the rind extract (extraction in Soxhlet apparatus and distillation).
Methanolic pith extract (MPE) was extracted from pith which was separated from seeds and soaked in 250 mL of methanol overnight.After filtration it was distilled to re move solvent and used for testing the antimicrobial activity.

Preparation of aqueous extracts
Aqueous rind extract (ARE) was prepared from 10 g of powdered rind soaked in 100 mL of distilled water for 24 hours and then filtered.The filtrate was concentrated and used for testing the antimicrobial activity.
Aqueous seed extract (ASE) and aqueous pith extract (APE) were prepared following the same procedure as for the rind extract.
Crude pomegranate extracts (CPE) was prepared using 10 g of powdered whole pomegranate soaked in 100 mL of distilled water for 24 hours.After filtration the extract was concentrated and used for testing the antimicrobial activity.

Total phenol content procedure
Various concentration samples were mixed with 2 mL of sodium carbonate.After 2 minutes, 0.5 mL of diluted Fo linCiocalciteu was added and incubated at the room tem perature for 30 minutes.The absorbance was measured using a spectrophotometer at 720 nm.Standard Gallic acid of concentrations ranging from 1050 µg and water blank were processed together.The total phenolic content was expressed as Gallic acid equivalents.

Microorganisms
The following bacterial strains were obtained from the American Type Culture Collection (ATCC) and the Na tional Collection of Industrial Microorganisms (NCIM) of the microbiology laboratory of Hubert Enviro Care Systems Pvt Ltd., Chennai, India: Staphylococcus aureus (ATCC 11105), Staphylococcus epidermis (ATCC 25619), Klebsiella pneumoniae (ATCC 9621), Pseudomonas aer uginosa (ATCC 25619) and Candida albicans (NCIM 3100).Each bacterial species was incubated in liquid culture dilutions at 37°C for 20 minutes to reach the logarithmic growth stage, and then measured to 0.5 Mc Farland dilution delivering the final concentration of ap proximately 105 CFU per mL.Then after, the agar plates with Methanolic and Aqueous pomegranate extracts were incubated overnight at 37°C.

Minimum inhibitory concentration
The antibacterial activity of natural products was stud ied using a micro dilution method.The inoculums were prepared by suspending bacterial and fungal strains in MüllerHinton broth.Extracts were dissolved in DMSO (10 % of the final volume) and diluted with culture broth to the concentration of 2 mg/mL.Further 1:2 serial dilu tions were performed by adding the culture broth to reach concentrations ranging from 2 to 0.0156 mg/mL; 100 μL of each dilution were distributed in 96well plates, as well as a sterility control and a growth control (containing cul ture broth plus DMSO without antimicrobial substance).Each test and growth control wells were inoculated with 5 μL of bacterial suspension (108 CFU/mL or 105 CFU/ well).The micro dilution trays were incubated at 36°C for 18 hours.The results were expressed in mg/mL.Posi tive controls were Streptomycin for bacterial strains and Amphotericin B for fungal strains.

Total polyphenol content
Total phenolic content of pomegranate is presented on Graph 1.The methanolic and aqueous extracts of pome granate pith had higher polyphenol content (805.6 g/meq of gallic acid) compared to other pomegranate part ex tracts.All parts of pomegranate had polyphenol content higher than 100 mg GAE/100 mL.

Minimum inhibitory concentration
MIC of all pomegranate extracts against different oral microorganisms obtained by serial dilution method is presented in Table 1.The highest MIC for Staphylococcus aureus showed MSE and ASE in the concentration of 12 mg/mL.The highest MIC against Staphylococcus epider mis was recorded by CPE in the concentration of 12 mg/ mL.The highest MIC against Pseudomonas aeruginosa and Candida albicans was found for MRE and ARE in the concentration of 12 mg/mL.All positive controls Strepto mycin (bacterial species) and Amphotericin B (Candida) were more efficient than the pomegranate extracts.

DISCUSSION
Considering wide variations in the total polyphenol con tent, foods are divided in two groups, high polyphenol content foods (>100 mg GAE/100 mL) and low polyphe nol content foods (<100 mg GAE/100 mL) [14].This clas sification is also adopted in the present study to classify total phenolic content of different pomegranate extracts.
Results showed that pomegranate is high polyphenol con tent food since it has polyphenol content higher than 100 mg GAE/100 mL.
The results of the current study are in agreement with Salgado et al. [15], who found higher antioxidant activity in pomegranate peels than in seeds and pulp.Due to the presence of phytochemicals such as phenolic compounds, tannins and flavonoids [16] pomegranate may have value as natural antioxidant with its high polyphenol content.
MIC is the minimum concentration of experimental extract required to inhibit the growth of particular organ ism.The antimicrobial activity of pomegranate has been investigated by Menezes et al. [17].Ethanolic, water, meth anolic and acetone extract of pomegranate showed strong antimicrobial activity in different investigations done on both Grampositive and Gramnegative nonoral bacte ria [17].A hydroalcoholic extract of pomegranate fruit was investigated for antibacterial effect on dental plaque microorganisms and was found to be effective against Sta phylococcus, Streptococcus, Klebsiella, and Proteus species, as well as Escherichia coli.Most likely ellagitannin and punicalagin are fractions responsible for pomegranate's antibacterial activity [17].
Thai researchers investigated the effect of biodegrad able chips impregnated with pomegranate pericarp on periodontal disease in 20 patients with gum pocket depths of 58 mm.Inflammatory markers interleukin1β (IL1β) and IL6 were significantly reduced from three to six months after intervention [18].Pomegranate rinsing was found to reduce activity of αglucosidase (sucrose degrading enzyme) in saliva as well as to increase the activity of ceruloplasmin (an antioxidant enzyme) [19].Pomegranate extract was also found to be effective in the treatment of denture stomatitis caused by C. albicans [20].Kakiuchi et al. [21] and Pereira et al. [22] demon strated specific antimicrobial action of pomegranate on dental biofilm bacteria, i.e., disturbance of polyglycan synthesis and adherence mechanisms of these organisms to dental surface.

CONCLUSION
All parts of pomegranate contain high polyphenol content and they are effective against some oral bacteria and C. albicans.Pomegranate can be a potential substitute for synthetic antibiotics against oral microorganisms.

UVOD
Is hra na ima va žnu ulo gu u spre ča va nju na stan ka obo lje nja usta i zu ba, uklju ču ju ći ka ri jes, ero zi ju zu ba, raz voj na ošte će nja, bo le sti oral ne slu zo ko že i, u ma njoj me ri, obo lje nja pa ro don ci ju ma [1].Ta ko đe, is hra na uti če na raz voj usne šu plji ne.Po sle di ce mo gu bi ti raz li či te u za vi sno sti od to ga ka da je po sto ja la nu tri tiv na ne rav no te ža.Ra no na sta la nu tri tiv na ne rav no te ža do vo di do na stan ka mal for ma ci ja [2], pa ta ko ne do sta tak vi ta mi na i mi ne ra la u fa zi pre za če ća uti če na raz voj bu du ćeg em bri o na, or ga no ge ne ze gla ve i vra ta, rast gor nje vi li ce, lo ba nje i li ca [3].Da bi se oču va lo oral no zdra vlje i ubla ži le bo le sti, neo p hod na je urav no te že na is hra na.U usnoj šu plji ni ži vi vi še od 700 vr sta mi kro or ga ni za ma [4,5].Mno gi unu tra šnji i spo lja šnji fak to ri uti ču na sa stav, me ta bo lič ku ak tiv nost i pa to ge nost oral ne mi kro flo re [4,5].Ta ko đe, do bro je po zna to da su mno gi pro iz vo di bilj nih eks tra ka ta, kao što su ta ni ni, ter pe no i di, al ka lo i di i fla vo no i di, no vi iz vo ri an ti mi krob nih sa sto ja ka ko ji po ma žu u bor bi pro tiv pa to ge na ot por nih na mno ge le ko ve [6].Po sto je tri glav na raz lo ga za što su bilj ni eks trak ti va žni: pr vo, far ma ko lo ške stu di je su po ka za le da mno ge bilj ke ima ju an ti mi krob na svoj stva; dru go, po sto je mno ge nus po ja ve po ve za ne s upo tre bom an ti bi o ti ka; i tre će, broj mi kro or ga ni za ma ot por nih na an ti bi o ti ke se po ve ća va [7].
Po li fe no li su naj ve ća gru pa fi to he mi ka li ja ko je su na đe ne u bilj ka ma, po seb no u vo ću, se men ka ma i li šću.Ovi sa stoj ci su ko ri sni u oču va nju zdra vlja jer do pu nju ju i po ma žu funk ci o ni sa nje vi ta mi na i en zi ma an ti ok si dan sa u ob ra ni od ok si da tiv nog stre sa uzro ko va nog po ve ća nom ko li či nom re ak tiv nih ki se o nič nih gru pa.Po li fe no li iz hra ne su za ni mlji vi zbog svog po zi tiv nog de lo va nja na ljud sko zdra vlje.Čest unos vo ća bo ga tog po li fe no li ma po ve zan je sa sma nje nim ri zi kom od raz vo ja mno gih bo le sti [8].
Eks trakt na ra se po ka zao ko ri snim u sma nje nju bro ja pa to ge nih bak te ri ja i ri zi ka od na stan ka zub nog pla ka, gin gi vi ti sa i pa ro don to pa ti je, ka ko je po ka za no u kli nič kim is pi ti va nji ma [9].Glav ni bi o ak tiv ni sa stoj ci na ra su po li fe no li, ko ji ima ju sna žna an ti ok si dant na svoj stva [10].Pu ni ca gra na tum, po zna ti ji kao nar, pri pa da fa mi li ji Pu ni ca ce ae [11].Sta blo na ra obič no ra ste 12-16 me ta ra u vi si nu i ima tr no vi te gra ne.Zre li plod na ra mo že bi ti i do 12,5 cm du ga čak, tam no cr ve ne je bo je, sa ko ža stom ko rom i ob li ka gra na te sa iz ra že nim ka lik som.Plod sa dr ži mno go se men ki (Arils) raz dvo je nih be lom mem bra no znom pe ri kar pom, gde je sva ka se men ka okru že na ma lom ko li či nom ki se log, cr ve nog so ka.Li sto vi su sjaj ni i du gač ki oko 7,6 cm [12].Pu ni ka la gin je glav ni po li fe nol ko ji ima an ti ok si dant na svoj stva u so ku na ra [13].
Cilj ove stu di je bio je da ana li zi ra uku pan sa dr žaj po li fe no la u eks trak tu raz li či tih de lo va na ra (se me na, sr ži, ko re) i si ro vog eks trak ta, te da utvr di mi ni mal ne in hi bi tor ne kon cen tra ci je (MIK) eks tra ka ta raz li či tih de lo va na ra pro tiv raz vo ja oral nih mi kro or ga ni za ma.

Priprema voća
Za ana li zu je obez be đen nar ko ji je ku pljen na lo kal noj pi ja ci u Če na ju, u In di ji.Vo će je pri pre mlje no ta ko što je naj pre na pra vljen rez na vr hu oko dr ške (ovaj deo je po znat kao kru na).Rez je išao kroz ko ru i bio je kru žnog ob li ka.Na kon ukla nja nja kru ne, se men ke su po sta le vi dlji ve.Po tom su na pra vlje na tri plit ka pro re za se če njem kroz ko ru i srž, od vr ha do dna vo ća.Ta ko je vo će po de lje no na tri de la.Ovi de lo vi su po to plje ni u hlad nu vo du.Se men ke su odvo je ne od ko že i sr ži i oce đe ne u ce dilj ki da bi se uklo ni li osta ci sr ži.

KRATAK SADRŽAJ
Uvod Pre va len ci ja bo le sti zu ba u In di ji je u po ra stu.Sve ve ći broj mi kro or ga ni za ma re zi stent nih na an ti bi o ti ke do veo je do raz vo ja mno gih al ter na tiv nih me to da le če nja, me đu ko ji ma je i fi to te ra pi ja.Ta ko se nar (Pu ni ca gra na tum Linn) po ka zao efi ka snim u le če nju raz li či tih in fek ci ja.Cilj ove stu di je bio je da iz me ri sa dr žaj po li fe no la i mi ni mal ne in hi bi tor ne kon cen tra ci je (MIK) raz li či tih de lo va na ra ko je spre ča va ju rast oral nih mi kro or ga ni za ma.Ma te ri ja li i me to de ra da Na kon pri pre me me ta nol nog i vo de nog eks trak ta se me na, sr ži i ko re, kao i si ro vog eks trak ta na ra, od re đen je uku pan sa dr žaj po li fe no la po mo ću Fo lin-Čo kal či te u o vog (Fo lin-Ci o cal ci teu) re a gen sa uz pri me nu gal ne ki se li ne kao stan dar da.MIK je od re đen za če ti ri bak te rij ske vr ste i jed nu glji vi cu.Re zul ta ti Me ta nol ni i vo de ni eks trakt sr ži su po ka za li vi sok sa dr žaj po li fe no la (805,6 g/meq gal ne ki se li ne) u od no su eks trak te dru gih de lo va na ra.Me ta nol ni i al ko hol ni eks trak ti se me na su bi li efi ka sni pro tiv Staphylo coc cus aure us, si ro vi eks trakt na ra bio je efi ka san pro tiv Staphylo coc cus epi der mis, dok su me ta nol ni i al ko hol ni eks trakt ko re bi li efi ka sni pro tiv Pse u do mo nas aeru gi no sa i Can di da al bi cans.Za klju čak Svi de lo vi na ra sa dr že vi sok sa dr žaj po li fe no la i ima ju an ti bak te rij ska i an ti glji vič na svoj stva.Nar mo že bi ti za me na za sin te tič ke an ti bi o ti ke pro tiv oral nih mi kro or ga ni za ma.Ključ ne re či: nar; oral ni pa to gen; po li fe no li; pu ni ka la gin; re zi sten ci ja

Priprema metanolnog ekstrakta
Me ta nol ni eks trakt ko re (MRE) je pri pre mljen se če njem ko re u sit ne kva dra ti će (po vr ši ne oko 5 mm 2 ) ko je su po tom osu še ne na 55°C to kom 24 sa ta i uskla di šte ne u do bro za tvo re noj tam noj ku ti ji do da lje upo tre be.Je dan uzo rak je imao 10 g osu še nog ma te ri ja la.Me ta nol ni eks trakt je do bi jen pri me nom eks trak to ra Sox hlet to kom osam sa ti.Na kon de sti la ci je, da bi se uklo nio ras tva rač, eks trakt je ko ri šćen za is pi ti va nje an ti mi krob ne ak tiv no sti.
Me ta nol ni eks trakt se me na (MSE) pri pre mljen je ta ko što su se men ke na kon su še nja na 55°C to kom 24 sa ta pod vrg nu te istom po stup ku kao i ko ra na ra (eks trak ci ja u apa ra tu Sox hlet i de sti la ci ja).
Me ta nol ni eks trakt sr ži (MPE) do bi jen je ta ko što je srž odvo je na od se men ki i pre ko no ći po to plje na u 250 ml me ta no la.Na kon fil tra ci je i de sti la ci je eks trakt je ko ri šćen za is pi ti va nje an ti mi krob ne ak tiv no sti.

Priprema vodenog ekstrakta
Vo de ni eks trakt ko re (ARE) je pri pre mljen ko ri šće njem 10 g ko re u pra hu ko ja je naj pre po to plje na u 100 ml de sti lo va ne vo de to kom 24 sa ta, a po tom pod vrg nu ta pro ce su fil tra ci je.Na kon kon cen tro va nja, fil trat je ko ri šćen za is pi ti va nje an ti mi krob ne ak tiv no sti.
Vo de ni eks trakt se me na (ASE) i vo de ni eks trakt sr ži (APE) su pri pre mlje ni na isti na čin kao i eks trakt ko re.
Si ro vi eks trakt na ra (CPE) je pri pre mljen ta ko što je 10 g pra ha do bi je nog od ce log na ra po to plje no u 100 ml de sti lo va ne vo de to kom 24 sa ta.Na kon fil tra ci je, eks trakt je kon cen tro van i ko ri šćen za is pi ti va nje an ti mi krob ne ak tiv no sti.

Određivanje ukupnog sadržaja fenola
Do bi je ni eks trak ti na ra po me ša ni su sa 2 ml na tri jumkar bo na ta.Po sle dva mi nu ta do da no je 0,5 ml raz bla že nog Fo lin-Čo kal či te u o vog (Fo lin-Ci o cal ci teu) re a gen sa, a sme sa je in ku bi ra na 30 mi nu ta na sob noj tem pe ra tu ri.Ap sorp ci ja je iz me re na po mo ću spek tro fo to me tra na 720 nm.Kao stan dard ko ri šće na je gal na ki se li na kon cen tra ci je od 10 do 50 µg, kao i vo da.Ukup ni fe nol ni sa dr žaj je iz ra žen kao ekvi va lent gal ne ki se li ne.

Mikroorganizmi
Bak te rij ske vr ste do bi je ne su iz Ame ri can Type Cul tu re Col lec tion (ATCC) i Na ci o nal ne ko lek ci je in du strij skih mi kro or ga ni za ma (NCIM) iz la bo ra to ri je za mi kro bi o lo gi ju Hu bert En vi ro Ca re Systems Pvt Ltd, Chen nai, In dia, i to: Staphylo coc cus aure us (ATCC 11105), Staphylo coc cus epi der mis (ATCC 25619), Kleb si el la pne u mo ni ae (ATCC 9621), Pse u do mo nas aeru gi no sa (ATCC 25619) i Can di da al bi cans (NCIM 3100).Sva ki mi kro or ga ni zam je u teč noj kul tu ri raz re đen i in ku bi ran na 37°C dva de set mi nu ta da bi do sti gao lo ga ri tam sku fa zu ra sta, a po tom me ren ko ri šće njem raz re đi va ča McFar land 0,5, da bi se do sti gla kon cen tra ci ja od oko 105 CFU/ml.Po tom su mi kro or ga ni zmi na aga ru in ku bi ra ni s me ta nol nim i vo de nim eks trak ti ma na ra na 37°C pre ko no ći.

Minimalna inhibitorna koncentracija
An ti mi krob na ak tiv nost je is pi ta na me to dom mi kro ra zre đi va nja.Naj pre su pri pre mlje ne su spen zi je is pi ta nih bak te rij skih vr sta i glji vi ce u Mi ler-Hin to no vom (Müller-Hin ton) bu jo nu.Te sti ra ni eks trak ti su ras tvo re ni u DMSO (10% od ko nač ne za pre mi ne) i raz bla že ni u bu jo nu u kon cen tra ci ji od 2 mg/ml.Do da va njem bu jo na do bi je na su se rij ska raz bla že nja u od no su 1:2, da bi se po sti gle kon cen tra ci je od 2 do 0,0156 mg/ml, a po tom je po 100 μl od sva kog raz re đe nja ras po re đe no u ko mo ri ce (u po su di sa 96 ko mo ri ca).Za kon tro lu ste ril no sti is pi tan je bu jon sa DMSO bez an ti mi krob nih eks tra ka ta.U testko mo ri ce i ko mo ri ce za kon tro lu ra sta ino ku li sa no je 5 μl su spen zi je mi kro or ga ni za ma (108 CFU/ml ili 105 CFU/ko mo ri ca).Ko mo ri ce su po tom in ku bi ra ne na 36°C to kom 18 sa ti.Re zul ta ti su iz ra že ni u mg/ml.Strep to mi cin je ko ri šćen kao po zi tiv na kon tro la za bak te rij ske vr ste, a am fo te ri cin B za glji vi cu.

REZULTATI Ukupan sadržaj polifenola
Ukup ni sa dr žaj po li fe no la na ra pri ka za n je na gra fi ko nu 1. Me ta nol ni i vo de ni eks trakt sr ži na ra ima li su ve ći sa dr žaj po li fe no la (80,56 g/meq gal ne ki se li ne) u od no su na eks trak te dru gih de lo va na ra.Svi de lo vi na ra su po ka za li sa dr žaj po li fe no la ve ći od 100 mg GAE/100 ml.

Minimalna inhibitorna koncentracija
MIK eks tra ka ta svih de lo va na ra pro tiv raz li či tih oral nih mi kro or ga ni za ma do bi je ni me to dom se rij skog raz re đi va nja pri ka za ne su u ta be li 1. Re zul ta ti po ka zu ju da su naj vi ši MIK za S. aure us ima li MSE i ASE u kon cen tra ci ji od 12 mg/ml.Naj vi ši MIK pro tiv S. epi der mis za be le žio je CPE u kon cen tra ci ji od 12 mg/ml.Naj vi ši MIK pro tiv P. aeru gi no sa i C. al bi cans ima li su MRE i ARE u kon cen tra ci ji od 12 mg/ml.Obe po zi tiv ne kon tro le strep to mi ci na (za bak te rij ske vr ste) i am fo te ri ci na B (za C. al bi cans) bi le su efi ka sni je od eks tra ka ta na ra.

DISKUSIJA
U od no su na raz li ke u ukup nom sa dr ža ju po li fe no la, na mir ni ce su po de lje ne u dve gru pe: na one s vi so kim sa dr ža jem po li fe no la (>100 mg GAE/100 ml) i ni skim sa dr ža jem po li fe no la (<100 mg GAE/100 ml) [14].Ova kla si fi ka ci ja je ta ko đe usvo je na u na šoj stu di ji za kla si fi ka ci ju ukup nog sa dr ža ja po li fe no la kod raz li či tih eks tra ka ta na ra.Re zul ta ti su po ka za li da nar pri pa da gru pi na mir ni ca ko je sa dr že vi so ku kon cen tra ci ju po li fe no la.Re zul ta ti na še stu di je su u sa gla sno sti sa stu di jom Sal ga da (Sal da go) i sa rad ni ka [15], ko ji su uoči li bo lju an ti ok si da tiv nu ak tiv nost u ko ri na ra ne go u se me nu i pul pi.Zbog pri su stva fi to he mi ka li ja, kao što su fe no li, ta ni ni i fla vo no i di u sr ži na ra [16], nar mo že bi ti zna ča jan pri rod ni an ti ok si dans.
MIK je naj ma nja kon cen tra ci ja eks pe ri men tal nih eks tra ka ta ko ja je po treb na da spre či rast od re đe nog mi kro or ga ni zma.An ti mi krob na efi ka snost na ra je ana li zi ra na u stu di ji Me ne ze sa (Me ne zes) i sa rad ni ka [17].Al ko hol ni, vo de ni, me ta nol ni i ace ton ski eks trak ti na ra su po ka za li iz ra že no an ti mi krob no dej stvo pro tiv Grampo zi tiv nih i Gramne ga tiv nih bak te ri ja [17].Vo de noal ko hol ni eks trakt na ra je po ka zao an ti bak te rij sku efi ka snost pro tiv mi kro or ga ni za ma zub nog pla ka Staphylo coc cus, Strep to coc cus, Kleb si el la, vr ste Pro te us, kao i Esche ric hia co li.Naj ve ro vat ni je je da su ela gi ta nin i pu ni ka la gin frak ci je od go vor ne za an ti bak te rij ska svoj stva na ra [17].
Taj land ski is tra ži va či su is pi ti va li efi ka snost bi o ra zgra di vih či po va im preg ni ra nih sa pe ri kar pom na ra u le če nju pa ro don to pa ti je kod 20 pa ci je na ta ko ji su ima li pa ro don tal ne dže po ve du bi ne 5-8 mm.Stu di ja je po ka za la da je iz me đu tri me se ca i šest me se ci na kon in ter ven ci je do šlo do znat nog sni že nja in ter le u ki na 1β (IL1β) i IL6 [18].Is pi ra nje usne du plje po mo ću so ka na ra sma nju je ak tiv nost αglu ko zi da ze (en zi ma ko ji raz la že sa ha ro zu) u plju vač ki, a ujed no po ve ća va ak tiv nost ce ru lo pla zmi na (en zim -an ti ok si dans) [19].Eks trakt na ra se po ka zao efi ka snim i u le če nju sto ma ti ti sa kod no si la ca pro te ze uzro ko va nog gljivicom C. al bi cans [20].Ka ki u či (Ka ki uc hi) i sa rad ni ci [21] i Pe re i ra (Pe re i ra) i sa rad ni ci [22] po ka za li su da nar ima an ti mi krob na svoj stva pro tiv bi o filmbak te ri ja ta ko što do vo di do po re me ća ja sin te ze po li gli ka na i nji ho vog pri a nja nja za čvr sta zub na tki va.

ZAKLJUČAK
Svi de lo vi na ra sa dr že vi sok sa dr žaj po li fe no la i de lo tvor ni su pro tiv od re đe nih bak te ri ja i C. al bi cans.Nar mo že bi ti za me na za sin te tič ke an ti bi o ti ke u le če nju od oral nih mi kro or ga ni za ma.