Original Article

Yonago Acta medica 2015;58:129–136

The Effect of Bamboo Leaf Extract Solution and Sodium Copper Chlorophyllin
Solution on Growth and Volatile Sulfur Compounds Production of Oral Malodor
Associated Some Anaerobic Periodontal Bacteria
Abir Majbauddin, Isamu Kodani and Kazuo Ryoke

Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan

ABSTRACTBLES or 0.015% SCCS, PI; 0.001% BLES or 0.015%
SCCS, FN; 0.002% BLES or 0.007% SCCS, PN; 0.005%
BLES or 0.007% SCCS).
Conclusion High concentrations of both BLES (0.16%)
and SCCS (0.25%) show superior inhibiting capability
on all four oral malodor associated periodontal anaerobes during testing, suggesting that these compounds
might have a beneficial effect on oral health care.

Background Bamboo leaf extract solution (BLES) andsodium copper chlorophyllin solution (SCCS) are known
for their anti-oxidant activities. Oral malodor is often
related with periodontal pathogens. The present study
was undertaken to investigate the anti-bacterial effect ofboth BLES and SCCS on anaerobic periodontal bacteria
producing oral malodorous volatile sulfur compounds(VSC).
Methods Porphyromonas gingivalis W83 (PG), Prevotella intermidai TDC19B (PI), Fusobacterium nucleatum ATCC25586 (FN) and Prevotella nigrescence
ATCC33563 (PN) were investigated as oral isolated
bacteria. VSC production ability of the oral strains wasinvestigated by gas chromatography. With serial dilutionof BLES or SCCS, the strains PG, PI, FN or PN were cultured anaerobically with AnaeroPack at 37 ℃ for 3 days.
For the determination of anti-bacterial action of BLES or
SCCS, the inoculum was cultured with original concentrations of BLES 0.16% (w/v) or SCCS 0.25% (w/v).
Results Gas chromatography exhibited that all strains,
PG, PI, FN and PN were responsible for producing ahigh range of H2S and a moderate range of CH3SH. Anti-bacterial effect of BLES or SCCS on the strains wasobserved. Inhibition of BLES or SCCS on the strains
was revealed as concentration dependent. BLES or
SCCS inhibited bacterial proliferation at higher concentrations (PG; 0.04% BLES or 0.03% SCCS, PI; 0.002%
BLES or 0.03% SCCS, FN; 0.005% BLES or 0.01%
SCCS, PN; 0.01% BLES or 0.015% SCCS). No viablebacterial colony observed at original concentration of
BLES 0.16% or SCCS 0.25%. Strain growth was eliminated from inhibition at lower concentrations (PG; 0.02%

Key words

anti-bacterial effect; bamboo leaf extract
solution; oral malodor; sodium copper chlorophyllin solution; volatile sulfur compounds
For hundreds of years, plants have proven to be a valuable source for biologically active compounds. Indeed,from ancient history some plant extracts have been used
to cure various diseases. In this paper, we focus on twoof these extracts, bamboo leaf extract solution (BLES)
and sodium copper chlorophyllin solution (SCCS),
which are literally eco-friendly as well as biodegradable.
BLES has been used particularly in Asian countries
as an herbal medicine. Scientists believe various BLES
compounds possess numerous pharmacological capabilities because of their anti-oxidant properties.1, 2 Yearsof research indicate that BLES possesses qualities thatfight against microbes, diabetes, cancers, inflammation, obesity, fatigue, and lipidomics, while helping toprevent cardiovascular disease.3 In traditional systemsof medicine, bamboo has been used from ancient times
in various therapeutic applications for treating stomach
heat, epilepsy, cough and phlegm, fainting and loss of
consciousness in feverish diseases and a variety of mental disorders that develop with aging.4 In Japan, the traditional kumazasa bamboo leaf has been used to protect
food from bacterial attack, such as sheets (called “chimaki”) to wrap sushi in or for packing rice balls. This isapart from its use for treating halitosis, body odor, stomatitis, hemorrhoids and healing wounds.5
On the other hand, SCCS can be extracted fromdifferent grass species as well as from bamboo leaves.6
SCCS also has a long history of traditional use in the

Corresponding author: Abir Majbauddin
ab_dnt@yahoo.com
Received 2015 July 2
Accepted 2015 July 24
Abbreviations: BLES, bamboo leaf extract solution; CFU, colony
forming unit; FN, Fusobacterium nucleatum ATCC25586; PG,
Porphyromonas gingivalis W83; PI, Prevotella intermidai TDC19B; PN, Prevotella nigrescence ATCC33563; SCCS, sodium
copper chlorophyllin solution; VSC, volatile sulfur compounds

129

A. Majbauddin et al.

medicinal arena, particularly in wound healing and odor
control.7 Based on available literature, SCCS possesses
potential anti-oxidative and anti-bacterial activity.8, 9 According to Goldberg,10 a water soluble chlorophyll solution was able to suppress malodor in a Vincent’s stomatitis patient after spraying it in the mouth and between
the teeth since this anti-odor ability is apparently due toits anti-oxidizing properties. Carballo et al.11 suggested
that SCCS can be effective as a gelatin film or coating to prevent food from microbial contamination. In
recent times both BLES and SCCS have become commercially available and consumers use them for various
purposes, especially as dietary supplements and foodpreservatives.12, 13 The consumption of chlorophyllin has
long been associated with potential health benefits andeven now, you can find many advertisements for manychlorophyll containing products, mostly dietary supplements. Considering that these benefits have alreadybeen researched, now the interest on SCCS and BLES is
spreading to dentistry.
To date, neither solution has ever been tested for
effectiveness against oral malodor associated anaerobic periodontal bacteria. Because of this, both of thesecompounds caught our attention as interesting enoughto implement a study to test their properties against oralmalodor associated anaerobic periodontal bacteria. We
felt sure that we had a strong basis for a good study as
the existence of a correlation between oral malodor andperiodontal bacteria has been well known for a longtime.14
Oral malodor, often called “bad breath,” defined
simply as a foul odor coming out of the mouth, is oneof the most common problems throughout the world. It
has become one of the major complaints made by patients when they visit the dentist after dental caries andperiodontal disease. The main etiology of oral malodor
is oral and non-oral in origin. However, approximately
80 to 90% of cases originate orally and are usuallyassociated with bacterial overgrowth on the dorsumof the tongue (stemming from poor oral hygiene), accumulation of food debris, bacterial plaque, gingivitis,
periodontosis, deep carious lesions, exposed necrotic
tooth pulp, pericoronitis, mucosal ulcerations, imperfectdental restorations, unclean dentures and factors causing
decreased salivary flow. Oral malodor is thought to becaused by volatile sulfur compounds (VSC) including
H2S, CH3SH and (CH3)2S produced by anaerobic periodontal bacteria.15 VSC are a family of gases which areprimarily responsible for halitosis, a condition in which
objectionable odor is present in the mouth; two membersof this family, H2S and CH3SH are strongly co-relatedwith intra-oral halitosis.16 Evidence suggests that anaero-

bic bacteria present in periodontal pockets of periodontosis are usually the primary cause of this condition.14
To find out possible etiological factors, multiple approaches require special diagnosis and investigation.
The present study, however, was undertaken to assess
the inhibiting capability of BLES and SCCS against oralmalodor associated anaerobic periodontal bacterium
and expected to have a major impact on suppressing oralmalodor.
MATERIALS AND METHODS
Obligatory anaerobic periodontal bacteria

All four of the following strains—Porphyromonas
gingivalis W83 (PG), Prevotella intermidai TDC19B(PI), Fusobacterium nucleatum ATCC25586 (FN), Prevotella nigrescence ATCC33563 (PN)—were purchasedfrom Professor Kazuyuki Ishihara (Tokyo Dental College, Tokyo, Japan).
Gas chromatography

Gas chromatography is usually applied to measurementof VSC of air in the mouth. This test is an efficient andhighly reproducible method for analyzing VSC and canbe measured selectively, sensitively and quantitatively.
Therefore, VSC production by strains PG, PI, FN and
PN were examined by gas chromatography.17 The system of VSC analysis includes oral chroma CHM-1 (Abimedical, Kawasaki, Japan), gas volume: 0.5 mL, measurement time: 8 min, detection unit: ng/10 mL, operating temperature limit: 10 to 30 ℃, and storage temperature limit: –20 to 60 ℃. A small pore on an agar plate
cap was first made and sealed with plastic tape before
culturing the strains. After achieving lawns of confluent
growth on the plate incubated at 37 ℃ for 3 days withAnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan),the air from the agar plate was aspirated through thesealed pore with a 1 mL gas-tight syringe. From the syringe, 0.5 mL air was discarded and 0.5 mL air directly
injected into the sample hole of the gas chromatograph.
After 8 min, results could be obtained. This procedure
was repeated five times for each bacterial strain.
Anti-bacterial agents

We used 0.16% (w/v) BLES and 0.25% (w/v) SCCS.
Both anti-bacterial solutions were provided by Tama
Biochemical (Tokyo). Components in BLES and SCCS
are shown in Table 1.
Culture media

Anaerobic culture media, BHK RS, PEA BHK and
BBE, were purchased from Kyokuto Pharmaceutical In130

Effect of BLES and SCCS on some oral anaerobes

ing procedure as BLES was done. The final series ofdilution was as follows: 200 μL of SCCS 0.25%, 0.12%,
0.06%, 0.03%, 0.015%, 0.007%, 0.003% and 0.001%.
After mixing with 100 μL of each bacteria, the suspension was uniformly inoculated onto the agar surface andincubated at 37 ℃ for 3 days with the AnaeroPack. The
experiment was repeated five times for each strain.

Table 1. The amounts of major inorganic and/or organic components in BLES and SCCS*
BLES (per 100g)

SCCS (per 100 g)

Na
260 mgNa
220 mg
Cu
72 mg
Cu
72 mg
K
50 mg
K
1 mg
Beta-carotene
400 μgMg
1.5 mgAlpha-tocopherol
1.4 mgFe
0.55 mg
Beta-tocopherol< 0.1 mgZn
0.18 mgGamma-tocopherol < 0.2 mg
Mn
0.017 mg
Delta-tocopherol
0.1 mgVitamin C< 1 mg
BLES, bamboo leaf extract solution; SCCS, sodium copper chlorophyllin solution.
*As for SCCS, components other than sodium copper chlorophyllin.

Culture for anti-bacterial assay (direct application)

At the beginning, bacterial suspensions corresponding
to 1.5 × 105 CFU/mL were first inoculated on the agar
plate and incubated for 1 day. After initial incubation,
the number of colonies were counted followed by 400
μL original concentration of BLES 0.16% or SCCS
0.25%, poured onto the 1 day incubated bacterial surface
and then again incubated at 37 ℃ for 3 days. Finally,colonies were counted. The experiment was performedin duplicate for each strain. In concentration dependent
test, inoculates were mainly mixed with several concentrations of BLES or SCCS and then cultured, therefore
the intention behind this particular experiment was tosee how bacterium react, when original concentrationof BLES or SCCS directly applied on them after 1 day
incubation. Furthermore, to determine the anti-bacterial
action, the inhibited non-colony zone from PN was inoculated on the agar plate with the help of needle inoculators and again incubated at 37 ℃ for 3 days.

dustrial (Tokyo). Prior to the anti-bacterial test selectionof appropriate culture medium was done on the basis ofproviding satisfactory bacterial growth, where BHK RS
showed the capability of producing good growth for all
bacterium compare to others medium, therefore BHK
RS medium was used throughout the experiments toassist all 4 strains anaerobic to grow well. The ingredients of BHK RS are follows: Polypepton 10.0 g, casein
pepton 10.0 g, yeast extract 5.0 g, glucose 1.0 g, sodium
sulfite 5.0 g, sodium chloride 5.0 g, vitamin K1 0.01 g,
hemin 0.01 g, sodium pyruvate 1.0 g, arginine 1.0 g, cysteine hydrochloride 0.3 g, agar 15.0 g, hemolytic rabbit
blood, defibrinated sheep blood, PH 7.0.

Statistical analysis

The data was presented as the mean ± SD after counting
the colony number. Statistical analysis was attained by
two-tailed paired-sample t-test with IBM SPSS software
version 21 (IBM, Armonk, NY).

Anaerobic culture

Three days of incubation at 37 ℃ with the AnaeroPack was performed.

RESULTS

Dilution liquid medium

Gas chromatography

Tryptic soy broth used as dilution liquid medium.

Gas-chromatographic evaluation is shown in Fig. 1. All
4 strains are capable of producing a high range of H2S,
particularly in strains PG, PI, and PN, where the amountof H2S exceeds 2 ng/mL, except for FN which is lessthan 2 ng/mL. Moderate release of CH3SH was detected
in all strains. The range of CH3SH in strains PG, PI, and
PN remained beyond 0.7 ng/mL to 1 ng/mL but for FN
which was below 0.5 ng/mL. (CH3)2S was only detectedin PI. However, this was a trace amount compared to
H2S and CH3SH. Regarding the gas-chromatographic
test in this study, strains PG, PI, FN and PN were revealed as strong candidates for producing oral malodor.

Anti-bacterial assay

To determine the anti-bacterial activity of BLES and
SCCS, the experiment was carried out by the serial twofold dilution method.18 Before testing, each bacterial suspension was prepared by 2 mL tryptic soy broth to givea starting concentration of approximately 1.5 × 105 CFU/
mL.
Two hundred μL of 0.16% BLES in tryptic soy broth
was followed by serial two-fold dilution with 200 μL
suspension each. Finally, 200 μL of BLES 0.16%, 0.08%,
0.04%, 0.02%, 0.01%, 0.005%, 0.002% and 0.001% wereobtained, respectively. Two hundred μL of 0.25% SCCS
was also diluted by tryptic soy broth and the same dilut131

A. Majbauddin et al.

Anti-bacterial effect of BLES and SCCS
Porphyromonas gingivalis W83

tions of 0.03% and 0.06%. SCCS were strongly inhibitory to PI (P < 0.001) in concentrations of 0.12% and 0.25%.
The anti-bacterial activity of SCCS on PI is lower thanthat of BLES.

The anti-bacterial activity of BLES on PG is shown in
Fig. 2a. PG growth was not inhibited in very low concentrations of BLES, 0.001% to 0.02% but graduallyinhibited as BLES was increased in a concentration
dependent manner. Statistically significant differencesof BLES with PG was revealed: P < 0.01 at 0.04%. Highinhibition of BLES with PG was revealed: P < 0.001 at
0.08% and 0.16%.
The anti-bacterial activity of SCCS on PG is shownin Fig. 2b, where SCCS inhibited PG in a similar manner as BLES in Fig. 2a. The inhibitory activities of
SCCS with PG were weak in very low concentrations of
SCCS, 0.001% to 0.01%. PG growth started to decrease
at 0.03%. Statistically significant differences of SCCS
with PG were revealed: P < 0.01 at 0.03% and 0.06%
and P < 0.001 at 0.12%. No bacterial growth was observed in the original concentration P < 0.001. The antibacterial activity of SCCS on PG is slightly higher thanthat of BLES.

Fusobacterium nucleatum ATCC25586

The anti-bacterial activity of BLES on FN shows in Fig.
2a. BLES greatly inhibited FN growth. The complete
inhibitory concentration of BLES on FN was higher at
0.08% (P < 0.001), which was the only activity as far as
we could see. Statistically significant values were foundP < 0.01 at 0.005% and 0.01%, and P < 0.001 at 0.02%
to 0.16%.
The anti-bacterial activity of SCCS on FN is shownin Fig. 2b. FN growth was gradually inhibited by increasing SCCS from 0.001%. Statistically significant
values were obtained at P < 0.01 in concentrations of
0.01% to 0.06%. It was highly inhibitory of SCCS on FN
in 0.12% and 0.25% P < 0.001. The anti-bacterial activity of SCCS on FN is lower than that of BLES.
Prevotella nigrescence ATCC33563

Prevotella intermidai TDC19B

The anti-bacterial effect of BLES on PN is shown in Fig.
2a. PN growth in lower concentrations of BLES, 0.001%
to 0.005% were slightly inhibited. PN growth gradually
decreased in higher concentrations of BLES. Statistically significant values were obtained: P < 0.01 at 0.01%
and 0.02%, and P < 0.001 at 0.04% to 0.16%.
The anti-bacterial effect of SCCS on PN is shownin Fig. 2b. PN growth in lower concentrations of SCCS,
0.001% to 0.007% were slightly inhibited. Statisticallysignificant values were obtained: P < 0.01 at 0.01% and0.03%, and P < 0.001 at 0.06% to 0.25%. The anti-bacterial activity of SCCS on PN is similar to that of BLES.
Anti-bacterial activity of SCCS with PG is higher
than that of BLES while it is lower with PI and FN.
However, anti-bacterial activity of SCCS with PN issimilar to that of BLES.

The anti-bacterial activity of BLES on PI is shownin Fig. 2a. Formation of PI colony started to decrease
with a lower concentration of BLES, 0.001%, and no PI
growth in the original concentration of 0.16% was observed (P < 0.001). Therefore, inhibition of PI growth ina concentration dependent manner was clearly observed.
The anti-bacterial activity of SCCS on PI shows in Fig.
2b. SCCS was weakly affected by PI growth inhibition
than BLES. In lower SCCS concentrations, 0.001% to
0.01%, PI growth was moderately inhibited.
SCCS inhibited PI growth (P < 0.01) in concentra30

PG
PI
FN
PN

ng/10 mL

25

20

Culture for anti-bacterial assay (direct application)

BLES (original concentration) inhibition on the 4 strains
is shown in Fig. 3a. After 1 day incubation, all four
strains showed similar growth and then after 3 days of
incubation with BLES 0.16%, PI, FN and PN were completely inhibited at the original concentration (P < 0.001);
however, PG survived with a fewer number of colonies (P< 0.01).
SCCS (original concentration) inhibition on the 4
strains is shown in Fig. 3b. All strains were inhibited at
0.25% SCCS compared with 1 day of incubated bacteria. Statistically significant values were obtained (P <0.001) for PI, FN and PN, (P < 0.01) for PG. It was noted

15

10

5

0

H 2S

CH 3SH(CH 3)2S

Gas-chromatography
Fig. 1. Investigation of VSC producing capability of four oral
strains (PG, PI, FN and PN), performed by gas chromatography.
Error bars represent mean ± SD for 5 time experiments. FN, Fusobacterium nucleatum ATCC25586; PG, Porphyromonas gingivalis W83; PI, Prevotella intermidai TDC19B; PN, Prevotellanigrescence ATCC33563.

132

Effect of BLES and SCCS on some oral anaerobes

10

a

BLES
PG

9

PI

8

CFU/plate/log

FN

7

PN

*

6

*

*

*

5

*

4

*

** **

**

3

**

**
**

**

2
1
0

Control 0.001

0.002

0.005

0.01

0.02

0.04

Concentration of BLES (w/v)

10

b

0.08

(%)

SCCS
PG

9

PI

CFU/plate/log

8

FN

*

7

**

6
5

PN
*

*

*

*
*

4

*
**

3

**

2

**
**
**

1
0

**
0.16

Fig. 2. Survival rate of PG, PI, FN
and PN with gradual concentrationsof BLES or SCCS. The bar charts
represent the CFU/plate/log. on, mean
± SD from 5 independent experiments. Control means of (1.5 × 105
CFU/plate/mL). The values **P <0.001 and *P < 0.01 are shown with
controls.
a: Survival rate of PG, PI, FN and PN
with BLES. b: Survival rate of PG,
PI, FN and PN with SCCS.
BLES, bamboo leaf extract solution;
CFU, colony forming unit; FN, Fusobacterium nucleatum ATCC25586;
PG, Porphyromonas gingivalis W83;
PI, Prevotella intermidai TDC19B; PN,
Prevotella nigrescence ATCC33563.

Control

0.001

0.003

0.007

0.01

0.03

0.06

Concentration of SCCS (w/v)

that BLES showed a relatively strong anti-bacterial effect compare to SCCS against all strains. Additionally,
when inhibited non-colony zone from PN was cultured,
the bacterial strain showed further proliferation (data notshown).

0.12

**
0.25

(%)

PG, PI, FN and PN by gas chromatography, results indicated all four species are capable of producing a highrange of H2S (around 2 ng/mL) and a moderate range ofCH3SH (around 1 ng/mL) except FN (below 0.5 ng/mL).
Yaegaki et al.17 demonstrated after evaluation of mouth
air from periodontosis patients indicated that H2S levelremains below 1 ng/mL and CH3SH remains around 4
ng/mL compare with healthy subjects. This is an opposite to what we found, but the difference could dependon both bacterial characteristics and the environment
in which they were grown. However, the mechanism of
VSC production in periodontal disease is complicatedand may also involve other clinical factors.17 Studies
found in the literature suggested that for the formation of
H2S and CH3SH, anaerobic bacterium PG, PI, FN and
PN are not only highly co-related but other anaerobic

DISCUSSION
Oral malodor emerging from the degradation of organic
substrates such as glucose, mucin, peptides and proteinspresent in saliva, crevicular fluid, oral soft tissues andretained debris and some microbial degradation products are volatile sulfur compounds (VSC).15 Among
VSC, H2S and CH3SH are the prominent elements oforal malodor associated with chronic periodontosis.19
Regarding our study, after evaluation of the gas from133

A. Majbauddin et al.

bacterium such as Actinobacillus actinomycetemcomitans, Tannerella forsythia and Treponema denticola also
play a vital role.20, 21 Our gas-chromatographic results
showed that among the four bacteria only PI can produce (CH3)2S. Another study agrees with this, showingthat (CH3)2S in the oral breath has a positive correlation with PI.21 Not only this, but VSC, especially H2S
and CH3SH, are referred to as highly toxic and not only
responsible for oral malodor, but may also contribute to
the pathogenesis of periodontosis.14
We have showed the anti-bacterial activity of BLES
and SCCS against anaerobic periodontal bacteria for
the first time in this study, demonstrated in a concentration dependent manner, where all 4 strains were highly
susceptible to the original concentration of the solution.
The anti-bacterial activity of BLES and SCCS has beenshown to be dependent on both solution concentrations
and bacterial species or strains against which they aretested. It is evident that the inhibitory action of BLES
and SCCS were enhanced by their components, namely;
Na, Cu and K, which are contained in both BLES and
SCCS. Among them, Na in the form of NaClO has beenused effectively as an anti-bacterial agent in endodontic
irrigation solutions and has a strong anti-bacterial effectagainst oral anaerobes PG, PI and PN.22 Besides that, Cu
also has an anti-bacterial effect against few multidrugresistant bacteria,23 and KI has been used in treatments

3ato clean root canals of mandibular molars because of its
anti-microbial activity.24 In addition, when BLES and
SCCS are introduced into an oral anaerobe environment,these solutions create an unfavorable environment for
the anaerobes and may act in living cells to break downcarbon dioxide and free oxygen that prevents anaerobic
bacterial respiration due to their anti-oxidant ability.
In this study, BLES exhibited a relatively higher
anti-bacterial activity compared to SCCS against PI and
FN but SCCS showed a stronger effect against PG, andPN which resulted in both solutions having a similareffectiveness. We believe these phenomena of difference and similarity may be defined by some component
differences among the solutions and individual bacterial characteristics, while Na, Cu and K are containedin both. SCCS contains additional components of, Mg,
Fe, Zn and Mn, all of these having anti-bacterial activity.25–28 Fe combined with Cu can delay anaerobic bacterial growth and Zn citrate also can decrease anaerobic
bacteria from oral cavity.26, 27 Consequently, it is difficult
to determine which of the identified compounds might
be among the most active constituents for BLES, which
is highly effective. Bamboo leaf have components likebeta carotene, alpha tocopherol, gamma tocopherol, allhaving strong anti-bacterial activity,29 moreover phenolic compounds that possessed by bamboo leaf itself also
proven to have strong bactericidal action.30 Therefore we

BLES
1 day incubation
without BLES

3

Additional 3 dayincubation with BLES

SCCS
1 day incubation
without SCCS

Additional 3 day-incubation
with SCCS

2.5

CFU/plate/log

CFU/plate/log

2.5

2

1.5

1

2

1.5

1

*
0.5

0

b

*
**

0.5

PG

**

PI

**

FN

**

0

PN

**
**

PG

PI

FN

PN

Fig. 3. Growth rate of four strains after 1 day incubation, followed by additional 3 days incubation with 0.16% BLES or 0.25%
SCCS. Error bars represent mean ± SD from duplicate experiments. The values **P < 0.001 and *P < 0.01 are shown with 1 day
incubation.
a: Growth rate of 4 strains with or without 0.16% BLES. b: Growth rate of 4 strains with or without 0.25% SCCS.
BLES, bamboo leaf extract solution; CFU, colony forming unit; FN, Fusobacterium nucleatum ATCC25586; PG, Porphyromonas
gingivalis W83; PI, Prevotella intermidai TDC19B; PN, Prevotella nigrescence ATCC33563; SCCS, sodium copper chlorophyllin
solution.

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Effect of BLES and SCCS on some oral anaerobes

hypothesized that these compounds may be the reason
for BLES more effective.
In this study, when either BLES or SCCS (original
concentration) was poured directly on a 1 day incubatedbacterial surface, it inhibited bacterial growth. However,further culturing of non-colony zone inoculates revealed
that bacterial strains can further proliferate, which might
indicate that both BLES and SCCS possess bacteriostatic ability. Highest concentration of BLES and SCCS,
eventually been able to inhibit four bacterial growth andproliferation entirely in concentration dependent assay,
therefore we used highest concentrations of BLES and
SCCS in direct application assay to see whether they caninhibit bacterial growth entirely like concentration dependent assay. After the original anaerobic-culture of PN
was treated with SCCS for 3 days, no growth zone on
the SCCS-containing agar were touched by a loop andwere incubated on a BHK RS plate without SCCS whereabundant colonies appeared for another 3 day anaerobic
culture (data not shown). This particular experiment was
mainly undertaken to find out whether the anti-bacterial
activity of BLES and SCCS are bactericidal or bacteriostatic.
To emphasize BLES and SCCS as an anti-bacterial
agent, resistance to several antimicrobial agents in anaerobic bacteria has been more frequently increased.
Particularly genera prevotella and porphyromonas havebecome increasingly resistant to many anti-anaerobic
agents such as penicillins and cephalosporins. Antimicrobial agents commonly used in the treatment of anaerobic infections are beta-lactam antibiotics (carbapenems),
metronidazole and beta-lactam compounds (ampicillin, amoxicillin, ticarcillin and piperacillin), the broadspectrum quinolones moxifloxacin and gatifloxacin havepotential to treat mixed aerobic and anaerobic infections,however resistance to these agents seems to increase
among several genera of anaerobic bacteria.31 In addition both BLES and SCCS can be regarded as safe andnon-toxic, study showed that maximum tolerated doseof bamboo leaf in both rats and mice was greater than
10 g/kg body weight and did not shown any significant
hematological, clinical, chemical and histopathologicalchanges, literally no adverse reaction was found when
leucopenia patients treated with SCCS made tablet with
40 mg dose.32, 33
In treating oral malodor, the first line of defense
should be to identify the cause and eliminate it, so it
is obvious that a reduction or inhibition of bacterial
growth on the dorsum of the tongue is most important.
Suppressing oral malodor could be done through botha mechanical and therapeutic approach including properly continued oral hygiene along with tooth brushing,

tongue scraping, and flossing combined with the use ofan anti-microbial mouth cleaning products, a numberof products being commercially available including
toothpastes and tongue gels, and mouthwashes whichcontain antimicrobial agents to reduce the number ofbacteria. However, many of these don’t have extended
effectivity.34 Chlorhexidine is considered the most effective anti-plaque and anti-gingivitis agent, although it has
some side effects such as brownish discoloration of thepellicle covering the teeth and tongue, alteration in taste,increased desquamation of oral mucosa and increased
calculus formation.34 Regarding anti-bacterial effects,
few chemicals might be beneficial to neutralizing oralmalodor, although these may include zinc chloride andsodium chloride.35 Products containing natural substances, usually classified as cosmetics, may improve
the patient compliance; probiotic micro-organisms inchewing gums or lozenges might restrain the re-growthof bacteria associated with oral malodor by preemptively
colonizing the oral cavity. To date, there are not so many
clinical studies investigating the clinical efficacy of oral
products containing probiotics.36
In conclusion, considering these results, BLES and
SCCS certainly have inhibiting effects on growth andproliferation of oral anaerobic bacteria PG, PI, FN and
PN, particularly against high concentrations, all bacterium showing superior susceptibility. In this context, theanti-bacterial activity of BLES and SCCS makes themstrong candidates for the development of a new antibacterial agent against bacteria causing oral malodor andassociated oral diseases.
Acknowledgments: We would like to thank Dr. Masumi Ueda,
former professor at the Tottori University College of Medical Care
Technology, for his precious advice and support in this study andProfessor Seiji Kageyama and Professor Shuhei Tomita for theirinvaluable suggestions and critical review of this research.
The authors declare no conflict of interest.

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