Siswomihardjo et al.

Int Chin J Dent 2007; 7: 27-29.

The difference of antibacterial effect of neem leaves and stick
extracts
Widowati Siswomihardjo, DDS, MSc, PhD,a Siti Sunarintyas Badawi, DDS, MSc, PhD,aMasahiro Nishimura, DDS, PhD,b and Taizo Hamada, DDS, PhDba

Department of Biomaterials, Faculty of Dentistry, Gadjah Mada University, Jogjakarta, Indonesia
Department of Prosthetic Dentistry, Graduate School of Biomedical Sciences, Hiroshima University,Hiroshima, JapanbPurpose: This study determined the antibacterial effect of ethanolic neem leaves and stick extract in inhibiting
the growth of Sreptococcus mutans.
Materials and Methods: Two different parts of neem, leaves and stick, extracts using ethanol were prepared at
10% and 20% (w/v: extraction powder/water) respectively. Each extractions were dropped on an MHA agar that
had been inoculated with Streptococcus mutans. Distilled water was used as the control. After 24 hours ofincubation, the inhibition diameters were measured. Collected data were analyzed by analysis of variance(ANOVA) followed by Least Significance Difference (LSD) at a 95% confinement level.
Results: The inhibition zone value of neem extracts on Streptococcus mutans showed that neem leaves extract
had less inhibition value than neem stick extract on all concentrations. The ANOVA showed that there weresignificant influence of neem extracts (p<0.001), neem concentrations (p<0.001), and neem extractconcentration (p<0.023) on Streptococcus mutans inhibition.
Conclusion: Neem leaves and stick ethanolic extracts had antibacterial effect on Streptococcus mutans. Neem
stick extract had higher antibacterial properties than the leaves extract. (Int Chin J Dent 2007; 7: 27-29.)
Key Words: antibacterial effect, neem extracts, Streptococcus mutans.

Introduction
The use of therapeutic plants has been beneficial to the oral health throughout the world for more thanthousands years. Over centuries, different parts of neem plant, stem bark, root, leaves, seeds, etc., have beenused in the Indian folk medicine. The advantage of traditional medicine is that it is less likely to form allergies
and side effects. Widespread use of antibiotics in dental practice gives microorganisms enhanced opportunities
for the development of resistance to a broad spectrum of antibiotics.1 Neem is one of the most widely researchedtropical trees as the source of therapeutic agents. The chemical compositions of neem extract have been
analyzed since twenty years before. Many active components of neem, azadirachtin, salannin, meliantriol, ornimbin etc., have been identified, and the most active ingredient is reported as azadirachtin.2 Clinical studies
have shown that the neem leaves extract decreased the dental plaque index and Streptococcus mutans andLactobacilli growth.3,4 An in vitro study has demonstrated that aqueous extract from Neem leaves inhibits
biofilm formation and adhesion in composite resin by Candida albicans.5

Also it has demonstrated thatformation of the bacterial plaque has been positively affected by aqueous neem stem bark extract.6 Manyexperiments have tested the aqueous neem extract. On the other hand, the methanolic extract of neem hasreported to have in vitro antiviral activity against group B coxsackieviruses7 or against Staphylococcus Aureus,
Escherichia Coli, Pseudomanas Aeruginosa, and Candida albicans.8 The extraction methods might affect theantibacterial efficiency. However, it has not yet well examined the antibacterial effects of ethanolic extract ofneem toward Sreptococcus mutans that has been recognized as the major organism involved in caries. Based onthese backgrounds, this study was aimed to compare the influence of ethanolic neem leaves and stick extract ininhibiting the growth of Sreptococcus mutans.

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Int Chin J Dent 2007; 7: 27-29.

Materials and MethodsExtract preparation
Neem leaves or sticks, 300 g, were selected, washed, cut into small pieces, and dried in an oven (45-50°C) for
3 days. Neem leaves or sticks were blended using a blender, then extracted using 96% ethanol for 10 hours. The
extraction was carried out using soxhlet instrument as described previously.8

Three doses of the dried

extractions, 20 g of extract in 100 mL distilled water (20%), 10 g of extract in 100 mL distilled water (10%), andno extract in 100 mL distilled water (control) were prepared.
Bacterial sensitivity test
The Streptococcus mutans sensitivity test was carried out using the agar diffusion disc technique.9 The
Streptococcus mutans were cultured in the MHA agar for 24 hours at 37˚C. Five colonies were transferred into 2mL of BHI. Its turbidity was compared to the Standard Brown III solution. The suspension (1 mL) was takenusing a micropipette and inoculated on the MHA agar petri dish. A sterile spreader was used to spread thesuspension evenly on the agar. Neem extracts (50 µL) at each concentration were dripped into the holes. Thepetri dish was incubated at 37˚C for 24 hours.
Inhibited zone measurement
The inhibited zone was measured as the area around the hole where no Streptococcus mutans was growing.
The required area was measured from the edge of the hole to the outer border of bacterial inhibition. The
diameter was measured using a sliding caliper with a precision of 0.01 mm. Each measurement was taken threetimes and the average of the three measurements for each zone was recorded.
Statistical analysis
Statistical analysis of the data was carried out using the analysis of variance (ANOVA). The ANOVA wasthen followed by post-hoc tests (Tukey-Kramer multiple comparison test).

Results
The average and standard deviations of the inhibited zones of Streptococcus mutans on MHA agar treatedwith neem extracts were shown on Table 1. The result showed that the antibacterial effects were significantly
different among control, neem leaves, and stick extracts (p<0.001, ANOVA). Post-hoc test (Tukey-Kramer)
showed that neem stick extract (at 20%) had significantly higher antibacterial property than the leaves extract(p<0.01). The neem stick extract had antibacterial properties as concentration dependent manner (p<0.01).
Table 1.

The inhibition zone value of neem extract toward Streptococcus mutans.

Extract concentration(%)

Neem leaves
MeanSD

Neem stickMeanSD0 (control)
1020

0.000
3.089 a3.857 a0.000
4.022 a, b
5.778 a, b0.000
0.661
0.211

0.000
0.713
0.408

NSNS
p<0.01

SD, Standard deviation. NS: Not significant between the properties of leaves and stick extracts.
a, significantly different from control (p<0.001); b, significantly different between different concentrations (p<0.01).

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Int Chin J Dent 2007; 7: 27-29.

Discussion
In this study, we have shown that the ethanolic neem stick extract had higher antibacterial properties than theleaves extract. Neem leaves had the active component of azadirachtin (1-3%), whereas the active component ofneem stick is tannin (6%).10 Both the azadirachtin and tannin belongs to the polyphenol group. The differencein the antibacterial effect was probably because of the different percentage in the active component of phenolgroups in both neem leaves and sticks. Neem stick extract possessed a wide spectrum of antibacterial action
against gram negative and gram positive microorganism.11 Hydrolyzable tannins, gallotannins from crude drugs
has an inhibitory activity against glucosyltransferase from Streptococcus mutans.12 Gallotannin is hardly soluble
in water but easily in ethanol. The reason that the neem stick extraction had higher antibacterial properties thanthe leaves extraction might be caused by the extraction solvent in this study differ from previous studies.5,6
Table 2 also showed that both extract and concentration of neem influence the inhibition zone value. The neem
leaves extract had less inhibition zone value comparing to the neem stick extract on the concentration of 10 and20% by ethanol extraction method. By this finding, it seems necessary to study further about the characteristicof the active components and their extraction methods which affect the antibacterial properties.

References
1. Ndukwe KC, Okeke IN, Lamikanra A, Adesina SK, Aboderin O. Antibacterial activity of aqueous extracts of selected chewing
sticks. J Contemp Dent Pract 2005; 6: 86-94.
2. Koul O, Isman M, Kethar C. Properties and uses of neem (Azadirachta indica). Can J Bot 1990; 68: 1-11.
3. Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel --a 6-week clinical study.
J Ethnopharmacol 2004; 90: 99-103.
4. Vanka A, Tandon S, Rao SR, Udupa N, Ramkumar P. The effect of indigenous Neem Azadirachta indica [correction of(Adirachta indica)] mouth wash on Streptococcus mutans and lactobacilli growth. Indian J Dent Res 2001; 12: 133-44.
5. Polaquini SR, Svidzinski TI, Kemmelmeier C, Gasparetto A. Effect of aqueous extract from Neem (Azadirachta indica A.
Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans. Arch Oral Biol 2006; 51:482-90.
6. Wolinsky LE, Mania S, Nachnani S, Ling S. The inhibiting effect of aqueous Azadirachta indica (Neem) extract upon bacterial
properties influencing in vitro plaque formation. J Dent Res 1996; 75: 816-22.
7. Badam L, Joshi SP, Bedekar SS. 'In vitro' antiviral activity of neem (Azadirachta indica. A. Juss) leaf extract against group B
coxsackieviruses. J Commun Dis 1999; 31: 79-90.
8. Okemo P, Mwatha W, Chhabra S, Fabry W. The kill kinetics of azadirachta indica A. Juss (Meliaceae) extracts on
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Afr J Sci Tech 2001; 2: 113-8.
9. Koreman E, Allen S, Dowell V, Sommers H. Color atlas and textbook of diagnostic microbiology. Vol. 2nd ed. California:Williams and Wilkins; 1983. p. 432-5.
10. Trewari DN. Monograph on Neem (Azadirachta indica). New Delhi: Int Book Distributor; 1992. p. 179.
11. Agarwal SK, K. DV. New medicinal properties of neem - a multipurpose farm forestry tree. J Indian Forestry 1995; 121: 1003.
12. Kakiuchi N, Hattori M, Nishizawa M, Yamagishi T, Okuda T, Namba T. Studies on dental caries prevention by traditionalmedicines. VIII. Inhibitory effect of various tannins on glucan synthesis by glucosyltransferase from Streptococcus mutans.
Chem Pharm Bull (Tokyo) 1986; 34: 720-5.

Correspondence to:Dr. Masahiro Nishimura
Department of Prosthetic Dentistry, Division of Cervico-Gnathostomatology
Graduate School of Biomedical Sciences, Hiroshima University
1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
Fax: +81-82-257-5684 E-mail: maakun@hiroshima-u.ac.jp
Received January 29, 2007. Accepted March 9, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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