ORIGINAL RESEARCH

Year : 2015 | Volume : 26 | Issue : 5 | Page : 477--482

Ant -m crob al eff cacy of All um sat vum extract aga nst Enterococcus faecal s b of lm and ts penetrat on nto the
root dent n: An n v tro study
Ourv nd J.S. B rr ng1, Ilum nada L V lor a2, Ph des Nunez3,
1 Department of Conservat ve Dent stry and Endodont cs, College of Dental Surgery, Un versal College of Med cal Sc ences, Bha rahawa, Ind a
2 Department of Endodont cs, Un vers ty of the East, Man la, Ph l pp nes
3 Department of Per odont cs, Un vers ty of the East, Man la, Ph l pp nes

Correspondence Address:
Ourv nd J.S. B rr ng
Department of Conservat ve Dent stry and Endodont cs, College of Dental Surgery, Un versal College of Med cal Sc ences, Bha rahawa
Ind a

Abstract
Introduct on: Sod um hypochlor te (NaOCl) has long been the most preferred root canal rr gant n endodont c treatment, but bes des be ng an effect ve ant -m crob al agent, t s h ghly
cytotox c. Thus, a search for an alternat ve herbal rr gant wh ch would be more b ocompat ble but equally effect ve led to th s study. A m: To assess the ant -m crob al eff cacy of garl c
extract (GE) aga nst Enterococcus faecal s b of lm and ts ab l ty to penetrate nto root dent n. Mater als and Methods: E. faecal s was cultured and treated w th the test agents - normal
sal ne, 5.25% of NaOCl, and the three d fferent concentrat ons of GE (10%, 40%, and 70%). The exper ment was done n four groups namely, 24-h Co-treatment group, 24-h b of lm
treatment group, 1-week b of lm group, and 3-week b of lm group. These groups were subjected to m crob al v ab l ty assay and fluorescence m croscop c analys s. The most effect ve
concentrat on of garl c (70%) was further tested and compared w th 5.25% NaOCl for ts dent n penetrat on property us ng 0.2% al zar n red under a fluorescence m croscope. Results:
The f nd ngs revealed that GE was able to d srupt as well as prevent the format on of b of lm produced by E. faecal s. All the concentrat ons of GE d splayed cons derable ant -m crob al
eff cacy where 70% concentrat on was most effect ve and exh b ted s m lar ant -m crob al eff cacy as 5.25% NaOCl. In terms of dent n penetrat on, no s gn f cant d fference was found
between GE and NaOCl. Conclus on: The results nd cate that GE has a potent al to serve as an alternat ve herbal root canal rr gant be ng an effect ve and b ocompat ble ant -m crob al
agent w th good dent nal penetrat on property.

How to c te th s art cle:

B rr ng OJ, V lor a IL, Nunez P. Ant -m crob al eff cacy of All um sat vum extract aga nst Enterococcus faecal s b of lm and ts penetrat on nto the root dent n: An n v tro study.Ind an J
Dent Res 2015;26:477-482

How to c te th s URL:
B rr ng OJ, V lor a IL, Nunez P. Ant -m crob al eff cacy of All um sat vum extract aga nst Enterococcus faecal s b of lm and ts penetrat on nto the root dent n: An n v tro study. Ind an J
Dent Res [ser al onl ne] 2015 [c ted 2020 Dec 16 ];26:477-482
Ava lable from: https://www. jdr. n/text.asp?2015/26/5/477/172041

Full Text
There are many requ s tes for endodont c treatment to succeed: Adequate cleans ng and proper shap ng of the canal followed by sat sfactory root canal f ll ng. The recommended
procedures for the b omechan cal preparat on of root canals are well-establ shed. Ex st ng l terature s replete w th stud es regard ng var ous techn ques of canal preparat on, from
establ sh ng the correct work ng length to the appropr ate shap ng of canals, such as ser al preparat on and crown-down techn ques. [1],[2]

One avenue of nvest gat on that also deserves much attent on s the search for endodont c mater als that w ll contr bute to further ncrease the success of root canal treatment. To th s
end, many researchers have stud ed the var ous types of newer f les, [3],[4] as well as d fferent types of gutta-percha, sealers and cements. Stud es regard ng root canal rr gants;
however, are fewer, wh ch may be due to the fact that sod um hypochlor te (NaOCl) has long been the gold standard and has been proven to be effect ve n d s nfect ng the canal as well
as d ssolv ng any remnants of pulp t ssue. [5],[6],[7]

Desp te the success of NaOCl as a root canal rr gant, t s st ll worth look ng nto poss ble alternat ves because t shows to be h gh tox c ty, result ng n necrot c effects when t comes n
contact w th normal t ssues. [8],[9],[10]

Consequently, the search for alternat ve products such as natural phytochem cals solated from plants used n trad t onal med c ne would be reasonable. [11]

One such plant product conta n ng natural phytochem cals s garl c. Garl c (All um sat vum) s one of the most extens vely nvest gated med c nal plants n use s nce anc ent t mes due to
ts ant -bacter al, ant -fungal, and ant -v ral propert es. [12] Its extract has been shown to have a w de spectrum nh b tory effect on the growth of var ous Gram-pos t ve and Gram-negat ve
bacter a. [13],[14],[15]

In th s study, the authors nvest gated the eff cacy of garl c (A. sat vum) extract to reduce the number of v able Enterococcus faecal s, to prevent the format on of b of lm produced by E.
faecal s and ts ab l ty to penetrate an ex st ng b of lm, along w th ts property to penetrate nto the dent nal tubules.

MATERIALS AND METHODS

Preparat on of garl c extract

Fresh natural garl c (A. sat vum) was obta ned from the Bureau of plant ndustry (Department of Agr culture), Man la. It was then blended n a ster l zed mortar and pressed w th gauze.
Th s extract was centr fuged at 12,000 rpm for 10 m n and then f ltered w th a 0.45-mm f lter to obta n raw garl c extract (GE) and stored at −20°C unt l use.

Culture of Enterococcus faecal s and treatment w th agents

E. Faecal s stra n (ATCC 47077) was noculated n bra n heart nfus on broth and ncubated overn ght at 37°C n a shaker ncubator. A standard E. faecal s bacter al suspens on was
prepared us ng McFarland 0.5 (1.5 × 10 8 CFU/ml). E. faecal s bacter al suspens on (100 μL) was d spensed nto four ster le flat-bottomed 96-well polystyrene m crot ter plates. In the f rst
plate, des gnated as the "co-treatment" group, raw GE was mmed ately added to E. faecal s (100 μL) suspens on to obta n 10%, 40%, and 70% concentrat ons and then ncubated at
37°C for 24 h. For the second, th rd, and fourth plates, E. faecal s suspens on was ncubated at 37°C for 24 h, 1-week, and 3 weeks, respect vely, to allow for the growth and format on of
b of lm. These were des gnated as the "24-h," "1-week," and "3-week" groups. The med um was replaced regularly to replen sh the nutr ents. After the allotted t me, the three rema n ng
groups (24-h, 1-week, and 3-week groups) were each treated w th 10%, 40%, and 70%, respect vely, GE n the same manner. For compar son, controls were prepared for all the plates
as follows: Pos t ve control (E. faecal s + normal sal ne [NS]), negat ve control (E. faecal s + 5.25% NaOCl). A total of three wells were noculated per concentrat on and per agent; hence,
all the samples were processed n tr pl cates. For all the exper mental groups, the b of lms were exposed to the agents for 10 m n after wh ch, the agents were carefully removed w th a
m crop pette. 100 μL ster le de on zed d st lled water was then added, after wh ch the m crob al v ab l ty assay and b of lm sta n ng procedures were performed.

To determ ne the amount of v able bacter a 10 μL of Presto Blue cell v ab l ty reagent (Inv trogen, USA) was added to all the wells n the m crot ter plates. The plates were ncubated at
37°C for 30 m n and then read at 570 nm us ng m cro plate reader (B otek, USA) to determ ne the color metr c data opt cal dens ty (O.D). The nh b t on ndex was then calculated us ng
the follow ng formula: Inh b t on ndex (%) = 100 - ([O.D sample/O.D control] × 100).

B of lm format on was conf rmed by SYPRO ® Ruby B of lm Matr x sta n (Inv trogen, USA) and observed under the fluorescence m croscope (Appl ed Spectral Imag ng, Israel). 10 μL of
sta n ng solut on was added gently onto the b of lm. The samples were ncubated for up to 30 m n at room temperature protected from l ght. They were then r nsed gently w th f lter
ster l zed water to remove all the sta n and v ewed under the fluorescence m croscope. All the tests were performed accord ng to the manufacturer's nstruct ons.

Assessment of dent n penetrat on

The approval from Inst tut onal Rev ew Board was taken before conduct ng the study (Ref: 0013/E/O/10/11). To assess whether GE could penetrate nto the dent nal tubules, the protocol
descr bed by Paragl ola and Franco, [16] w th sl ght mod f cat ons, was followed. Br efly, 10 recently extracted human s ngle canal teeth were decoron zed to standard ze the lengths. The
work ng length was establ shed by nsert ng a s ze 10 K-type f le nto each canal unt l t was seen through the ap cal foramen then retract ng t by 0.5 mm. The canals were shaped w th
n ckel-t tan um rotary nstruments (Protaper, Dentsply Ma llefer, Sw tzerland) follow ng the usual protocol, w th s ze F3 as the last f le used at the work ng length.

The spec mens were d v ded nto two groups (f ve n each). The f rst group was rr gated w th 3 mL of 5.25% NaOCl labeled w th 0.2% al zar n red wh le the second group was rr gated
w th 3 mL of 70% GE labeled w th 0.2% al zar n red at each nstrument change w th a 30-G needle at 5 mm from the work ng length. Smear layer removal was ach eved after rr gat on n
both groups w th 3 mL of 17% EDTA (Pulpdent, USA) followed by 3 mL of ster le sal ne. A f nal r nse of each canal was performed by us ng 5 mL of al zar n labeled w th 5.25% of NaOCl
and 70% GE n each group, respect vely.

After dry ng the canals, each spec men from the both groups was hor zontally cut nto three 1-mm th ck sect ons at 1, 3, and 5 mm from the apex, des gnated as Sect on 1, Sect on 3, and
Sect on 5, respect vely. The sect ons were then bonded onto glass sl des and were ground w th wet s l con carb de papers to approx mately a 40 μm th ckness. The sl des were exam ned
w th a fluorescence l ght m croscope at ×100 w th a wavelength of 540 to 570 nm. Images from all the spec mens were evaluated by three bl nded operators. The follow ng set of scores
was used to assess the penetrat on of the rr gant solut on nto the dent nal tubules [F gure 1]: "1"-m nor (penetrat on n <50% of dent nal tubules) and "2"- major (penetrat on n ≥50% of
the dent nal tubules).{F gure 1}

Data obta ned were analyzed us ng ANOVA followed by mean compar son to compare the b of lm format on and d srupt on between the control and the exper mental groups and among
the exper mental groups us ng SPSS Vers on 16 (SPSS for W ndows, Ch cago, SPSS Inc.). Ch -square w th the two-s ded L kel hood rat o test and one-s ded F sher's exact test were
done to check the dent n penetrat on. The level of s gn f cance was establ shed as P < 0.05 for the stat st cal tests.

RESULTS

V able m croorgan sm count

The mean values of the data gathered for the v able m croorgan sm count of the f ve treatment groups at all the t me ntervals revealed that negat ve control, 40% and 70% GE d splayed
cons derable nh b t on of the bacter al growth. 70% GE and negat ve control d splayed s m lar results w th no s gn f cant d fference between them (P = 1.0) wh le the effect of 10% GE
was s m lar to the pos t ve control [F gure 2].{F gure 2}

Ev dence of b of lm format on for the control groups

B of lm format on, as assessed by the mod f ed SYPRO Ruby sta n was expressed n relat ve fluorescence un ts (RFUs) read at 620 nm. A h gh RFU value s nterpreted as the presence
of b of lm format on and conversely, a low RFU value means that there s l ttle or no b of lm format on. The pos t ve control (E. faecal s + NS) of 3-week b of lm group showed a th ck,
dense, and clustered b of lm format on as noted by the very h gh fluorescence values when compared to the pos t ve control of other b of lm groups, wh le the negat ve control (5.25%
NaOCl + E. faecal s) d splayed no b of lm format on w th low RFU values [Table 1].{Table 1}

Ev dence of d srupt on of b of lm format on for the treatment groups

For all the groups, t was observed that 10% GE exh b ted sl ght d srupt on n b of lm format on as compared to the pos t ve control. 40% GE showed a substant al d srupt on of b of lm
when compared to the pos t ve control (P < 0.05). Whereas, no b of lm format on was observed w th 70% GE as ev dent from the s gn f cantly smaller RFU value as compared to the
pos t ve control (P < 0.01). No s gn f cant d fference was found between 70% GE and negat ve control groups (P = 0.939) [F gure 3].{F gure 3}

Results for the fluorescence values of the treatment groups are summar zed n [Table 1]. Taken all together, the results nd cated that there was ncreas ng d srupt on of E. faecal s b of lm
format on as the concentrat on of the GE was ncreased. It can be seen from [F gure 4] that NaOCl and 70% GE had the lowest fluorescence values wh le the pos t ve control (E. faecal s
+ NS) had the h ghest fluorescence value.{F gure 4}

Penetrat on of garl c extract nto the dent nal tubules

[Table 2] shows the cross-tabulat on d splay ng major and m nor dent n penetrat on of both the treatment groups n Sect ons 1, 3, and 5. The result revealed that GE had a sl ghtly h gher
dent n penetrat on than 5.25% of NaOCl but the d fference was not s gn f cant. Th s was conf rmed by both the two-s ded L kel hood rat o test (P = 0.063) and one-s ded F sher's exact
test (P = 0.070), wh ch showed that there was no s gn f cant d fference n dent n penetrat on for the spec mens rr gated w th GE and NaOCl.{Table 2}

DISCUSSION

Several nvest gat ons have stud ed the ant -m crob al effects of GE; however, there s very l m ted nformat on about ts ant -m crob al eff cacy aga nst E. faecal s b of lm formed n root
canals and ts penetrat on nto the root dent n.

In the present study, t was found that 40% and 70% concentrat on of GE has s gn f cant ant -m crob al act v ty aga nst E. faecal s b of lm, w th 70% concentrat on be ng as potent as
5.25% of NaOCl n reduc ng the number of E. faecal s as well as n prevent ng and remov ng the bacter al b of lm. Th s s n l ne w th a study conducted by Borhan-Mojab et al., n 2012,
where 40% and 70% concentrat on of GE were found to be effect ve n s gn f cantly reduc ng the total sal vary m crob al populat on. [17] Another study done by Zakar a 2004,
demonstrated that the aqueous GE was effect ve aga nst an array of Gram-pos t ve and Gram-negat ve pathogens. [18] W th regards to ts use n endodont cs, a study done by Abu and
Sawsan n 2001, suggested that fresh m nced garl c solut on can be useful as an ntracanal med cat on as t nh b ted the growth of E. faecal s and Pseudomonas aerug nosa, α-hemolyt c
Streptococc , and Streptococc pyogenes.[19]
The m n mum nh b tory concentrat on (MIC) of GE on E. faecal s growth as determ ned by Lee et al., s 128 mg/ml (12.8%). [20] Th s expla ns the l m ted effect of 10% concentrat on of
GE (sub-MIC) n reduc ng the number of v able E. faecal s as well as ts b of lm.

It s worth ment on ng that careful cons derat on must be taken to preserve the effect v ty of the GE n ts method of preparat on. In th s study, garl c bulbs were crushed and the extracts
taken and f ltered w thout subject ng them to any exposure to h gh temperature dur ng the ent re procedure to preserve all c n wh ch s the most mportant ant -m crob al component of
garl c and to el m nate the poss b l ty of nact vat on of any of ts mportant components. Th s effect ve procedure of preserv ng all c n from be ng deact vated has been conf rmed by a
study conducted by Chavan et al. n 2010 wh ch reported that many therapeut c propert es n garl c are destroyed when subjected to heat. [21] It has been recogn zed that all c n, the
unstable compound formed by the enzymat c act on of all nase s nact vated at a temperatures above 65°C. [22]

One of the mportant propert es of NaOCl as a potent root canal rr gant s ts ab l ty to penetrate nto the dent nal tubules. Several stud es have been done to check and analyze the
dent n penetrat on ab l ty of the d fferent concentrat ons of NaOCl. Accord ng to a study conducted by Zou et al., 6% NaOCl d splayed the h ghest dent n penetrat on. [23] In the present
study, t was seen that 70% concentrat on of GE exh b ted good dent nal penetrat on s m lar to that of 5.25% NaOCl.

The present f nd ngs suggest that based on the demonstrated capab l t es, GE may prov de benef ts as an herbal root canal rr gant n prevent ng E. faecal s b of lm format on ns de the
root canals and may effect vely penetrate nto the root dent n. Lack of suff c ent n v vo stud es; however, proh b ts ts cl n cal pract ce recommendat on at the present t me. Further cl n cal
nvest gat ons for standard zat on and preparat on of the rr gant conta n ng th s ant -m crob al agent for the prevent on of bacter al b of lm format on n the root canal s proposed to conf rm
the eff cacy of GE for the r treatment. Further stud es may also look nto the poss b l t es of mask ng the strong odor of garl c extract so as to be of pract cal use n the cl n cal sett ng.

F nanc al support and sponsorsh p

N l.

Confl cts of nterest

There are no confl cts of nterest.

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