Phenolic antibacterials from Piper betle in the prevention of halitosis

Abstract

Piper betle L. (Piperaceae) leaves which are traditionally used in India and China in the prevention of oral malodor was examined by bioassay-guided fractionation to yield allylpyrocatechol (APC) as the major active principle which showed promising activity against obligate oral anaerobes responsible for halitosis. The biological studies with APC indicated that the potential to reduce methylmercaptan and hydrogen sulfide was mainly due to the anti-microbial activity as established using dynamic in vitro models.

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Phenolic antibacterials from Piper betle in the prevention of halitosis
Niranjan Ramji
a,
*,Nivedita Ramji
a
, Ritu Iyer
b
, S. Chandrasekaran
c
a
The Procter & Gamble Company, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
b
The Procter & Gamble Hygiene and Health Care Limited, P & G Plaza, Cardinal Gracias Road, Chakala, Andheri East, Mumbai 400099, India
c
401/402 Saurabh, 39 Swastik Park, Chembur, Mumbai 400071, India
Received 1 December 2001; received in revised form 1 July 2002; accepted 6 July 2002
Abstract
Piper betle L. (Piperaceae) leaves which are traditionally used in India and China in the prevention of oral malodor was examined
by bioassay-guided fractionation to yield allylpyrocatechol (APC) as the major active principle which showed promising activity
against obligate oral anaerobes responsible for halitosis. The biological studies with APC indicated that the potential to reduce
methylmercaptan and hydrogen sulfide was mainly due to the anti-microbial activity as established using dynamic in vitro models.
#2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Piper betle ; Antibacterial; Allylphenols; Halitosis; In vitro screening
1. Introduction
Halitosis, also commonly known as bad breath or
oral malodor, arises from the microbial degradation of
proteins (especially those that contain cysteine and
methionine), peptides and amino acids that are present
in the saliva, gingival cervical fluid or food retained on
teeth. The degradation products which are perceived as
foul smelling are the volatile sulfur compounds (VSCs)
such as methylmercaptan, hydrogen sulfide and volatile
fatty acids. Amongst the VSCs, methylmercaptan has
been regarded as the major contributor to halitosis
because of its very low odor threshold. Organisms
responsible for the production of VSCs include proteo-
lytic obligate anaerobes, especially the gram-negative
species that colonizes the subgingival plaque and
dorsum of the tongue (Kleinberg and Codipilly, 1995).
Piper betle L. (Piperaceae) leaves have been traditionally
used in India and China in the prevention of oral
malodor. The practice in India is to chew the leaves
alone or with areca nut and other spices such as
cardamom, clove and cinnamon, which act as ‘‘breath
fresheners’’ and help in the prevention of halitosis.
In our search for potent leads for the prevention of
oral malodor from natural sources, the methanol extract
of the fresh leaves of P. betle was subjected to in vitro
tests. These in vitro models included plate and broth
minimum inhibitory concentration (MIC) assays, bio-
film assay, saliva chip model and a conductometric
method developed in-house to evaluate methylmercap-
tan and hydrogen sulfide released by pure obligate oral
anaerobes and saliva samples. The spectrum of the
anaerobes studied consisted of Fusobacterium nucleatum
ATCC 25586, Porphyromonas gingivalis ATCC 33277
and Peptostreptococcus anaerobius ATCC27337, which
are the organisms known to be responsible for the
production of VSCs in the oral cavity. Besides these,
pooled saliva consisting of a heterogeneous microbiota,
which could also play an important role in causing
malodor was also studied in the biological models.
2. Methods
2.1. Extraction of plant material
The betel leaves were collected in Maharashtra State,
India, in January 1998. Voucher specimens were identi-
fied and were deposited in our herbarium (PG-Pb-0100).
Fresh betel leaves (1.2 kg) were extracted with methanol
* Corresponding author
E-mail address: ramji.n@pg.com (N. Ramji).
Journal of Ethnopharmacology 83 (2002) 149 
/152
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(3.0 l) and the aqueous methanol extract was concen-
trated under reduced pressure at 35 8C. This concen-
trated extract was successively partitioned into ether,
ethylacetate and aqueous fractions. The percent yield of
the ether, ethylacetate and the aqueous fractions based
on fresh raw material was 61.3, 1 and 36% (w/w),
respectively.
2.2. Fractionation and isolation of active principles
The ether fraction was subjected to bioassay-guided
fractionation using column chromatographic purifica-
tion (Silica gel 100/200 mesh) technique. The fractions
eluting with hexane, hexane containing 5% ethylacetate,
yielded three pure compounds which were identified to
be eugenol (1), chavibetol acetate (2) and allylpyroca-
techol monoacetate (3). All these three compounds
showed very little activity in the in vitro models. The
fraction eluting with hexane containing 10% ethylacetate
was found to be the most active. This fraction was
purified to yield allylpyrocatechol (4) (yield, 40.0% (w/
w) on raw material). The compounds 1/4were char-
acterized by spectral analysis (UV, IR,
1
H NMR and
MS) and found to be identical in every respect with
those reported earlier in the literature (Philip, 1984).
Furthermore, the acetylation of APC with acetic anhy-
dride and pyridine at room temperature affords APC
monoacetate (3). This was identical in all respects (TLC,
GC and IR analysis) with the isolated compounds (Fig.
1).
2.3. Bioassays
To assist the bioassay-guided fractionation, the fol-
lowing biological screens were employed.
2.3.1. MIC studies
Preliminary in vitro anti-microbial screens included
the MIC assays using the conventional agar plate
dilution and broth microdilution method (Shapiro et
al., 1994).
2.3.2. Biofilm assay
Any promising lead showing low MIC values were
subjected to the biofilm assay, which was designed to
mimic bacterial biofilms in the oral cavity. A 48-h-old
film prepared in 24-well tissue culture plate was
subjected to 1 min rinse with the treatment solution.
The treated film was reincubated in an anaerobic broth
for an additional period of 48 h. In the case of pure
culture biofilms, reincubation was carried out in Wilkins
Chalgren Anaerobic broth (WCB; Oxoid; CM643) while
in the case of saliva biofilms, WCB containing non-
sporulating anaerobe selective supplement (Oxoid;
SR107B) was used to select non-sporulating anaerobes
from a mixed population in the saliva sample. The
optical density of each well was measured at 490 nm and
the percent efficacy of a test solution was calculated with
respect to water control set. The biofilm assay provided
a more dynamic method to generate results which could
be extrapolated to evaluate the performance of any test
solution as a mouth rinse.
2.3.3. Methylmercaptan and hydrogen sulfide (VSC)
assay
Anovel in-house conductometric method was devel-
oped to quantify the levels of methylmercaptan and
hydrogen sulfide released from biological samples. The
indirect rapid automated bacterial impedance technique
(RABIT) from Don Whitley Scientific Ltd., UK, was
aptly modified for this purpose by using a sodium
methoxide bridge to trap methylmercaptan and hydro-
gen sulfide liberated from biological samples. In this
method, the methylmercaptan (CH
3
SH) and hydrogen
sulfide (H
2
S), which is liberated from the bacterial
culture/saliva sample upon incubation at 37 8C, are
trapped in the pre-aged sodium methoxide (NaOCH
3
)/
agar bridge which results in the formation of the sodium
salts of methylmercaptan (NaSCH
3
), hydrogen sulfide
(Na
2
S) and methanol (CH
3
OH). As both NaSCH
3
and
Na
2
Shave a lower conductivity than NaOCH
3
, there is
a decrease in conductance with the evolution of VSCs
from the biological sample. Hence a sharp and contin-
uous reduction in conductance is expected in the case of
faster VSC-releasing sample. These conductance
changes are quantifiable and the assay method was
validated. The evaluation performed using RABIT
involves the following steps:
a) 700 ml of 9.8 mg ml
1
NaOCH
3
prepared in 1%
agar was introduced at the base (in between the
electrodes) of the RABIT cell and the agar was
allowed to solidify before plugging the cells. The
bridges were aged overnight in the dark before the
assay.
Fig. 1. (1)R
1
/H, R
2
/CH
3
;(2)R
1
/CH
3
,R
2
/COCH
3
;(3)R
1
/
H, R
2
/COCH
3
;(4)R
1
/H, R
2
/H.
N. Ramji et al. / Journal of Ethnopharmacology 83 (2002) 149
/152150

b) Test the active principles at various concentrations
prepared in Witley’s anaerobic broth G5 0006
(supplemented with haemin and menadione) and
inoculated with F. nucleatum (3/10
8
cfu ml
1
); for
pooled saliva, a dilution of 1:5 was made with
maximum recovery diluent fluid of 2 ml sample of
each was introduced in the borosilicate tubes and
placed in the preaged RABIT cells.
c) The instrument was set up to the following para-
meters: test duration /24 h; detection criteria/10
ms; time resolution/6 min; temperature/37 8C.
The cells were loaded after system stabilization and
the test was started.
d) At the end of test duration, the absolute conductiv-
ity value was noted at 0.5, 4, 6, 8, 10, 12, 16 and 20 h
of test run. A total change in conductivity (T
change, DT) for each time point was calculated
with respect to the 30 min conductivity reading
which is considered as the starting point in the VSC
release kinetics.
e) A graph of DTand RABIT run time was plotted for
each test product and the overall release rate for a
period of 20 h was calculated from the area under
the curve (AUC), which represent the value for each
product. The AUCs of the active principles were
compared to their respective controls and their %
inhibition calculated by the formula:
/% Inhibition fAUCcontrol AUCactive
AUCcontrol
g100:/
2.3.4. Saliva chip model
The saliva chip model provided a method to study the
effect of test rinses on an immobilized plaque film
produced on hydroxyapatite chips (synamels). A 24-h-
old plaque matrix was given three 1 min treatments with
the test solutions. In between each treatment, the
synamel chips were reincubated in pooled saliva sample
under anaerobic conditions. 3 h after the last treatment,
the chips were evaluated for bacterial viability, using
densitometric measurements (O.D. read at 490 nm) and
VSC levels, according to the indirect conductometric
method mentioned above.
3. Results
The results of the screening are shown in Table 1.
Chlorhexidine was used as a positive control for all the
models. The % inhibition stated is the inhibition over
suitable solvent controls. The in vitro bioassays indi-
cated that only the ether fraction was active.
4. Discussion
The initial screening of the methanol extract of P.
betle leaves at 500 ppm showed an activity comparable
to 5 ppm of chlorhexidine in both the broth and plate
dilution assays. Partitioning of the methanol extract into
the ether, ethylacetate, aqueous methanol fractions was
done to further identify the active constituents. The
biofilm assay, saliva chip model, and the VSC assays
indicated that all the activity resided in the ether
fraction. The major constituent of the ether fraction
was identified to be APC which represents 80% (w/w) of
the ether fraction. This ether fraction showed an activity
quite comparable to APC in the biofilm assays (both
against anaerobes and pooled saliva) and VSC assays
(using F. nucleatum and pooled saliva samples). The
APC in the saliva chip model for anti-microbial activity,
and the VSC assay using pooled saliva, showed a slight
increase in activity over ether fraction. These results
clearly suggest that the reduction in the VSC production
by the oral anaerobic bacteria examined herein is largely
due to the anti-microbial activity of APC.
Table 1
In vitro % inhibition of P. betle extracts and APC
Sample tested Bioassay
Plate dil Broth dil Biofilm/ana Biofilm/pss Salivachip/VSC Salivachip/AM VSC/Fn VSC/pss
P. betel methanol extract (0.05%) 71 86
Chlorhexidine (0.0005%) 100 84 100 100
P. betel ether fraction (0.5%) 82 93 51 32
P. betel ether fraction (0.05%) 100 93
Chlorhexidine (0.5%) 88 98
Chlorhexidine (0.05%) 98 78
APC (0.5%) 89 94 71 31
APC (0.05%) 100 93
Plate dil, plate dilution assay; broth dil, broth dilution assay; biofilm/ana, biofilm assay against anaerobes; biofilm/pss, biofilm assay against
pooled saliva; salivachip/VSC, saliva chip model for the reduction in VSC production; salivachip/AM, saliva chip model for anti-microbial activity;
VSC/Fn, reduction in VSC formation from F. nucleatum; VSC/pss, reduction in VSC formation by pooled saliva sample.
N. Ramji et al. / Journal of Ethnopharmacology 83 (2002) 149
/152 151

Though the allylphenols from P. betle (leaf) have
been reported earlier, this is the first report which
establishes that these constituents exhibit strong anti-
microbial effects against obligate oral anaerobes,
thereby providing potential leads in the control of oral
malodor. The P. betle ether extract and APC could thus
be used in the prevention of halitosis in addition to the
prevention of periodontal infection caused by the
colonization of oral anaerobic bacteria.
References
Kleinberg, I., Codipilly, M., 1995. The biological basis of oral malodor
formation. In: Rosenberg, Mel (Ed.), Bad Breath: Research
Perspectives. Ramot Publishers, Tel Avive University, pp. 13 
/39.
Philip, H.E., 1984. Identification of fungicidal and nematocidal
components in leaves of Piper betle (Piperaceae). Journal of
Agriculture and Food Chemistry 32, 1254 
/1256.
Shapiro, S., Meier, A., Guggenheim, B., 1994. Oral Microbiology and
Immunology 9, 202 
/208.
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/152152