Indian J.Sci.Res. 7 (1): 342-347, 2014 ISSN: 0976-2876 (Print)
ISSN: 2250-0138(Online)
1Corresponding author
IN VITRO ANTIMICROBIAL EFFECTS OF AQUEOUS EXTRACTS OF CAESALPINIA
SAPPAN LINN. DERIVATIVES AGAINST ORAL PATHOGENS
DR. HAMED KERAMATa1, DR. ALI MOADDABIb AND DR. ARSALAN RANJBARIc
abcDDS Resident of Oral Medicine of Shahed University
ABSTRACT

Dental caries is a biofilm-related oral disease, and various antimicrobial agents have been developed for the prevention of dental diseases; however, many bacteria show resistance to existing agents. In this study, Sappan Lignum (the dried heartwood of Caesalpinia sappan L.)were evaluated for antimicrobial activity against five common oral bacteria as a screen for potential candidates for the development of natural antibiotics. Aqueous extracts of Sappan Lignum were tested for activity against Enterococcus faecalis, Actinomyces viscosus, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sanguis grown in brain heart infusion (BHI) broth. A broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A disk diffusion assay was performed by inoculating bacterial cultures on BHI
agar plates with paper disks soaked in each of the medicinal herb extracts. The aqueous extracts of Sappan Lignum demonstrated antimicrobial activity against the five types of pathogenic oral bacteria. The present study is confirmed the antimicrobial activity of the extract of Sappan Lignum against all five species of oral bacteria strains. These results suggest that certain herbal medicines with proven antimicrobial effects, such as Sappan Lignum, may be useful for the treatment of dental diseases.

with proven antimicrobial effects, such as Sappan Lignum, may be useful for the treatment of dental diseases.
KEYWORDS: antimicrobial activity, dental caries, Sappan Lignum, oral bacteria
Major dental diseases in the world are dental
caries and periodontal disease, both of which are caused
by various bacteria in the oral cavity. Periodontal disease
is one of the most common health problems in the human
communities (1) and dental caries is still a common
disease among children and adolescents (2). Many
developed countries have shown a marked decrease in
the prevalence of dental caries in children over the past
decades, however, in many other developing countries
caries prevalence has been increased(3). Gingivitis is one
of the most common forms of periodontal disease (4) and
around 100% of people aged 17 to 22 have gingivitis in
different degrees. Dental plaque, a biofilm of
microorganisms on tooth surface, plays an important role
in the development of caries and periodontal disease (5).
The accumulation and metabolism of bacteria on teeth
and implants surfaces are considered the primary cause of
caries, gingivitis, periodontitis, periimplantitis and breathe
(6). The accumulation of plaque on teeth is a highly
organized and ordered sequence of events (7). Cariogenic
bacteria and periodontopathic bacteria are present in
dental plaque as biofilms (8). Essentially, all oral bacteria
possess surface molecules that foster some type of cell-tocell
interaction (6). Only a few specialized organisms,
primarily streptococci are able to adhere to oral surfaces
such as the mucosa and tooth structure (7). Mutans
streptococci can colonize the tooth surface and initiate
plaque formation by their ability to synthesize
extracellular polysaccharides from sucrose, using
glucosyltransferase (9, 10).This sucrose dependent
adherence and accumulation of cariogenic streptococci is
critical to the development of pathogenic plaque (10). All
Streptococcus mutans serotypes such as Streptococcus
sobrinus (serotypes d, g and h) have been shown to have
significant potential to cause caries, but because of their
significant genetic and biochemical differences, they
should not be referred as simply as the single species S.
mutans. S. mutans and lactobacilli are acidogenic and
acid uric bacteria and seem to be the primary organisms
associated with caries in humans. S. mutans are most
strongly associated with the onset of caries, whereas
lactobacilli are associated with active progression of
cavitated lesions (7). Bacterial attachment to preexisting
plaque is studied by examining the adherence between
different bacterial strains (co-aggregation). One of the
best characterized interactions is the adherence of
Actinomyces viscosus through surface fimberiae to
polysaccharide receptor on cells of Streptococcus
sanguis (6). These types of interactions are thought to be
of primary importance in the colonization of the
periodontal environment (6). The further accumulation of
plaque around the gingival and subgingival region may
lead to a shift in its microbial composition from
streptococcus-dominated to a larger number of
Actinomyces spp. and an increased number of capnophilic
and obligatory anaerobic bacteria, such as Porphyromonas
KERAMAT ET AL.: IN VITRO ANTIMICROBIAL EFFECTS OF AQUEOUS EXTRACTS OF CAESALPINIA SAPPAN…
Indian J.Sci.Res. 7 (1): 342-347, 2014
gingivalis(11). Both streptococci and actinomycetes
which are facultative anaerobes and seem to be involved
in root caries and periodontal disease, respectively(12,13),
and doubling times for microbial populations during the
first 4 h of development are less than 1 h (6).
Consequently, these two groups of primary colonizers are
taught to prepare a favorable environment for secondary
colonizers, which have more fastidious growth
requirements. The microorganisms primarily considered
secondary colonizers fell into the green (includes
Eikenella corrodens), orange, or red complexes (6). E.
corrodens, a fastidious, slow growing, gram negative and
rod shaped bacteria that is part of the normal human oral
flora, has been isolated from a variety of infections
associated with human oral flora (14). This organism is
implicated as a human periodonto-pathogen and may also
cause extra oral infections (15). Recent advances in
microbiology and host defense studies allow clinicians to
couple conventional mechanical therapy with locally and
systemically delivered antimicrobial and host modulation
agents (6). Mechanical procedures such as root planning
is hard and takes usually more than one visit schedule and
can cause wearing of enamel (16); moreover, improved
understanding of the infectious nature of dental disease
has dramatically increased interest in chemical methods
of plaque control and holds great promise for advances in
disease control and prevention. Chemical plaque control
has been shown to be effective for both plaque reduction
and improved wound healing after periodonta surgery;
moreover it can augment mechanical plaque control
procedures (6). Antimicrobial agents against oral
microorganisms, especially those contributing to sub and
supra gingival biofilm formation, play an important role
in the prevention of dental caries, and periodontal disease
(17). Since some chemical materials including
Chlorhexidine can cause brown staining of the teeth
(16,6), tongue and silicate and resin restorations transient
impairment of taste perception, toxic effects on
connective tissues, dryness and soreness of oral cavity
(16), allergic reactions in patients (18) and oral
desquamation in children, use of herbal agents can be
a useful alteration. Herbal products have been used since
ancient times in folk medicine, involving both
eastern and western medical traditions (17). Many
plants and plant-derived antimicrobial components are
used in folklore therapeutics for the treatment of
periodontal disorders and for the purposes of oral hygiene
(18). Some have been evaluated for possible use in
modern medicine, while thousands of other potentially
useful plants have not been tested (18). During the last
two decades, the development of drug resistance as well
as the appearance of undesirable side effects of certain
antibiotics has lead to the search of new antimicrobial
agents mainly among plant extracts with the goal to
discover new chemical structures which overcome the
foregoing disadvantages (19-21). A wide range of
antimicrobial agents and herbal products are added to
dentifrice and mouth rinsing solutions with the aim of
preventing carries or biofilms formation (17).
Traditionally herbal medicines have been used to treat
infectious diseases since ancient times (22). Most oral
diseases are due to bacterial infections, and medicinal
plants are well known to exert considerable
antimicrobial activity against many microorganisms,
including the bacteria responsible for dental caries (23).
Furthermore, the natural phytochemicals isolated from
herbal medicines could offer effective alternatives to
antibiotics and represent a promising approach to the
prevention and treatment of dental caries and other oral
infections (24). Screening for herbal medicines effective
against oral bacteria is the required first step in the
identification of natural phyto-chemicals that could be
used as antimicrobial substances (22). Caesalpinia
sappan Linn, a traditional plant used widely in oriental
medicine. The plant extracts were found to be a good
source of secondary metabolites, vitamins and metals.
The extracts were further tested against certain human
pathogenic microbes. The methanol and ethyl acetate
extracts of the heartwood was found to be effective
against certain pathogenic microbes. In recent years, the
extract of Sappan Lignum (the dried heartwood of
Caesalpinia sappan L.) has been found to be a potential
immunosuppressive agent. The reported main phenolic
com- pounds in Sappan Lignum were divided into to four
structural sub-types: i.e. brazilin, chalcone, protosappanin
and homisoflavonoid. Among the protosappanin
derivatives, such as protosappanin B and
isoprotosappanin B, 10-O-methyprotosappanin B and 10-
O-methylisoprotosappanin B, as well as protosappanin E1
and protosappanin E2 occur as pairs of epimers.
Meanwhile, the homisoflavonoid epimers sappanol and
episappanol, 4- O-methylsappanol and 4-Omethylepisappanol,
3'-O-methylsappanol and 3'-Omethylepisappanol
were successively isolated along with
a new compound, a 3-benzylchroman derivative 3'-deoxy-
4- O-methylepisappanol (25). Considering the medicinal
importance of C. sappan, a study on the efficacy of C.
sappan extracts on human pathogenic bacteria and fungus
KERAMAT ET AL.: IN VITRO ANTIMICROBIAL EFFECTS OF AQUEOUS EXTRACTS OF CAESALPINIA SAPPAN…
Indian J.Sci.Res. 7 (1): 342-347, 2014
has been made anticipating to develop antibiotic in future.
Considering that only a few studies have been reported on
the in vitro effect of Sappan Lignum extracts against oral
pathogens and a great demand in dentistry for new and
better substances to inhibit or suppress bacteria and
biofilm formation, improve the quality of dental
treatment, and facilitate some dental procedures; this
study have been designed to evaluate the in vitro
antimicrobial activity of aqueous extract of Sappan
Lignum on some oral micro-organisms.
METHODS
Identification and preparation of medicinal herbs
extracts
Sappan Lignum used in this study were
purchased from the Medicinal Herbs Association.
Identification of all Sappan Lignum was verified by
Department of Pharmacy, Tehran University of Medical
Sciences. Sappan Lignum was extracted by heating in
water of 8 to 10 times of the herb weight for 3 hours at
115 ◦ C. After boiling, the extract was filtered using
standard testing sieves (150 m) and freeze-dried to a
powder. A 50 mg sample of powdered herbal medicine
was dissolved in 1 mL of distilled water and stored at −20
◦ C before use.
Microorganisms and growth conditions
Enterococcus faecalis, Actinomyces viscosus,
Streptococcus salivarius, Streptococcus sanguis and
Streptococcus mutans were purchased from the Pasture
Institute of Iran. Five types of strains were incubated in
brain heart infusion (BHI) broth and BHI agar at 37 ◦ C in
the presence of 5% CO2.
Determination of MIC and MBC of aqueous extract of
Sappan Lignum
Plate dilution method was used for
determination of the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) of
Sappan Lignum extracts on mixture of five kinds of oral
bacterial strains. For MIC and MBC, the bacteria were
incubated for 20 hours at 37 ◦ C in 5% CO2. To determine
MIC, the extract concentration was diluted two- fold from
5000 g/mL to 80 g/mL and the inoculums of 1 × 104
CFU/mL were used. The lowest concentration of the
herbal medicine extracts that inhibited the growth of the
organism, corresponding to an inhibition of 99% of the
inoculums, was considered as the MIC. To determine
MBC, we used a variant on the agar dilution method. The
inoculation spots with no visible growth were cut and top
down 3L of bacterial culture broth on BHI agar plate. The
lowest concentration of herbal medicine extract that
yielded no growth on the agar (99.9% killed) was defined
as MBC.
Agar diffusion assay
The antibiotic sensitivity profile of five kinds of
oral bacteria was determined on assay plate including
inoculums of 1 × 104 CFU/mL on the top layer of the
BHI agar plate. A sterile paper disk (8 mm) was soaked
with extracts of herbal medicines, so that each disk was
impregnated with 312-5000 g of Sappan Lignum extract
per disk. The plates were then incubated for 20 hours at
37 ◦ C in 5% CO2. The antibacterial activity was
evaluated by measuring the diameter (mm) of the
inhibition zone.
RESULTS
Sappan Lignum has strong activity against oral
bacteria
Aqueous extract of Sappan Lignum
demonstrated the strong antimicrobial activity, inhibiting
the growth of all oral bacteria examined. Agar plates
spread with oral bacteria were treated with a series of
dilutions, including 312, 625, 1250, 2500, and 5000g/mL
of Sappan Lignum. Most concentrations of Sappan
Lignum produced inhibition zones in all tested oral
bacteria, with the highest concentration, 5000g/mL, of
Sappan Lignum producing inhibition zones for mixture
culture of E. faecalis, S. salivarius, A. viscosus, and S.
sangui.
KERAMAT ET AL.: IN VITRO ANTIMICROBIAL EFFECTS OF AQUEOUS EXTRACTS OF CAESALPINIA SAPPAN…
Indian J.Sci.Res. 7 (1): 342-347, 2014
Figure 1: Disk diffusion assay of Sappan Lignum extract against oral bacteria. Inhibition zone formed according to
the concentration of the Sappan Lignum extract was indicated in mm. Control, 312g/mL, 625g/mL, 1250g/mL, 2500
g/mL and 5000g/mL (clockwise) respectively
DISCUSSION
The result of this study showed that aqueous
extract of Sappan Lignum, had strong antibacterial
activity against E. faecalis, S. salivarius, A. viscosus, and
S. sanguis, respectively. Dental disease is one of the most
prevalent public health concerns. The problems caused by
dental caries affect all age groups, and treatment is both
expensive and labor-intensive (26). Dental caries and
periodontal diseases are infectious diseases caused by
common oral bacteria; therefore, controlling or even
reducing the levels of these causative pathogens, such as
S. mutans and E. faecalis, is a key step in the prevention
and treatment of these diseases (27) Dental caries is a
common oral disease caused by many cariogenic
microbes, including Lactobacillus spp., Streptococcus
spp., and Actinomyces spp., which usually form plaque
biofilms on the tooth surfaces (26). Dental plaque is
initially synthesized by the glucosyltransferase from S.
mutans, and oral microorganisms then colonize and
accumulate in this water-insoluble glucan layer. The
viridans streptococci S. salivarius, S. sanguis, and S.
mutans were the most representative human cariogenic
bacteria included in the present study; however, these
species are also moderately resistant to antibiotics (24).
Actinomyces spp. is involved in early plaque
development on tooth surfaces and contributes to root
caries and periodontal infections. E. faecalis is an
opportunistic pathogen that is frequently isolated from
asymptomatic and persistent endodontic infections,
especially from the failed root canals undergoing
retreatment (28). E. faecalis is a better survivor than other
root canal microbes, being able to resist various harsh
conditions such as bile salts and starvation as well as
many antibacterial agents (29). The characteristics of
these oral bacteria determine the antimicrobial agents that
can be used for the prevention and treatment of dental diseases.
In our study, aqueous extracts of Sappan Lignum
were prepared and evaluated for their antimicrobial
activities against five species of oral bacteria. Sappan
Lignum has traditionally been used as a red dyestuff and
also as a herbal medicine to treat inflammation or improve
blood circulation (30,31). The antimicrobial activity of
Sappan Lignum against S. mutans has been reported by
scientist (32). However, we widely confirmed the
antimicrobial activity of Sappan Lignum about
periodontal diseases as well as dental caries. In our study,
the aqueous extract of Sappan Lignum showed strong
antimicrobial activity against all five species of oral
bacteria. The MIC values of Sappan Lignum against E.
KERAMAT ET AL.: IN VITRO ANTIMICROBIAL EFFECTS OF AQUEOUS EXTRACTS OF CAESALPINIA SAPPAN…
Indian J.Sci.Res. 7 (1): 342-347, 2014
faecalis and A. viscosus were especially low relative to
those of the other herbal medicines. Further, Sappan
Lignum extract had strong activity against oral bacteria in
the agar diffusion assay; this activity was more
pronounced for Streptococcus spp. than for other species
such as E. faecalis and A. viscosus. Therefore, we have
provided primary data showing that Sappan Lignum
extract could be a potential treatment for both dental
caries and periodontal diseases. In conclusion, we
evaluated the antimicrobial activities of aqueous extract
of Sappan Lignum against oral bacteria. Aqueous extract
of Sappan Lignum showed the strong antimicrobial
activity against all of the bacteria tested. Therefore, these
results suggest that herbal medicines with proven
antimicrobial effects, such as Sappan Lignum, may be
useful for the treatment of dental diseases.
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