Antimicrobial activity of frankincense of Boswellia serrata

Abstract

This work describes the in-vitro screening of anti microbial activity of frankincense
of Boswellia serrata. Different concentrations (25, 50, 75 and 100 mg /ml) was
evaluated for the investigation of antimicrobial efficacy using Gram positive
(Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumonia) and Gram
negative (E.coli, Klebsiella pneumonia, Enterobacter aerogenes, Pseudomonas
aeruginosa and Proteus vulgaris) microbes. Inhibition halos were evaluated and
compared with antibiotic Ciprofloxacin (5µg/ml) as positive control. DMSO was
used as a negative control. Results demonstrated significant antimicrobial activity.
In this assay, extracts of frankincense showed antimicrobial activity comparable
with standard and can be used in combating the bacterial infested diseases caused
by the studied bacterial strains.

Full text

Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1101
1095
Original Research Article
Antimicrobial activity of frankincense of Boswellia serrata
Shaik Mannur Ismail1, Sudheer Aluru2, KRS Sambasivarao1 and Bhaskar Matcha2*
1Department of Biotechnology, Acharya Nagarjuna University, Guntur, India
2Division of Animal Biotechnology, Department of Zoology, S.V University, Tirupati, India
*Corresponding author
A B S T R A C T
Introduction
Plant antimicrobials tender prospective
agent to deal with the hazard of biological
warfare (Gibbons, 2008). Consideration to
the sighting of novel plant antimicrobials
must be paid in this new era of
chemotherapeutic healing of infection by
using plant-derived principles and on
discovery of new antiinfective agents
(Cowan, 1999). Herbal preparations can
supplement other systems of medicine for
the treatment of diseases caused by bacteria
(Archana and Abraham, 2011). In fact,
efforts are going on to identify and isolate
secondary metabolites from plants as
prospective modulators of bacterial
resistance (Stavri et al., 2007).
Taxonomic status of Boswellia serrata
Boswellia serrata Roxb. ex Colebr.
(Burseraceae) is a deciduous moderate to large
sized branching tree that grows abundantly in
the seshachalam hill valleys.The
morphological characters have crown
spreading and flat, bark greenish, ashy grey,
ex-foliating in thin flakes. Leaves apically
clustered, imparipinnate. Leaflets 14-26, thin-
coriaceous, oblong-lanceolate, entire or
crenate, obtuse or subacute, secondary nerves
more than 16 pairs. Flowers pinkish white, in
little branched panicles. Sepals and petals 5-7
each. Stamens 10-16, inserted below disc;
disc annular. Ovary 3-locular; ovules 2 per
locule, pendulous. Fruits trigonous, brown,
pyrenes 3, heart shaped, each one seeded.
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 3 Number 10 (2014) pp. 1095-1101
http://www.ijcmas.com
Keywords
Boswellia
serrata,
Frankincense,
Antimicrobial
Activity,
Ciprofloxacin
This work describes the in-vitro screening of anti microbial activity of frankincense
of Boswellia serrata. Different concentrations (25, 50, 75 and 100 mg /ml) was
evaluated for the investigation of antimicrobial efficacy using Gram positive
(Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumonia) and Gram
negative (E.coli, Klebsiella pneumonia, Enterobacter aerogenes, Pseudomonas
aeruginosa and Proteus vulgaris) microbes. Inhibition halos were evaluated and
compared with antibiotic Ciprofloxacin (5µg/ml) as positive control. DMSO was
used as a negative control. Results demonstrated significant antimicrobial activity.
In this assay, extracts of frankincense showed antimicrobial activity comparable
with standard and can be used in combating the bacterial infested diseases caused
by the studied bacterial strains.

Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1101
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B. glabra Roxb., B. serrata Roxb. var.
glabra (Roxb.) Benn. are the synonyms. B.
serrata vernacularly (Telugu) known as
Guggilum, Anduga, Dhupamu,
Guggiladhpuam. Commonly known as
Salai, White dammar, ‘Indian olibanum’,
‘Indian frankincense’, ‘dhup’ and ‘salai’ or
‘salai guggul’ (Siddiqui, 2011).
Distributed commonly in lower hill slopes of
Tirumala and Talakona of Seshachalam hill
ranges. Flowering and Fruiting occurs in the
season of March-August. Traditional usage
of Gum is diuretic, diarrhoea, dysentery,
stomachic, cardiac diseases, cough,
haemorrhage, dyspnoea, polyuria,
leucorrhoea, oligospermia, urinary troubles,
piles, ulcers, burns (Madhava chetty et al.,
2013).
When incisions are made in the trunks of the
Boswellia serrata trees to produce exuded
gum (Oleo gum-resin), which appears as milk
like resin. The resin hardens (solidification)
into orange-brown gum resin known as
frankincense or olibanum. There are numerous
species and varieties of frankincense trees,
including Boswellia serrata in India.
The resins of Boswellia serrata have been
used for the treatment of rheumatoid arthritis
and other inflammatory diseases (Banno,
2006) such as Crohn's disease (Langmead,
2006) in traditional medicine of many
countries. The anti-inflammatory activity has
been attributing to the resin's ability in
regulating immune cytokines
production (Chevrier, 2005) and leukocyte
infiltration (Sharma et al., 1988; Singh and
Atal, 1986).
Fig.1 A. Flowering and Fruiting of Boswellia serrata B. Tree Habitat
C. Crude Frankincense resin

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Boswellia serrata extract also exhibits anti-
bacterial and anti-fungal activities
(Weckesser,2007). Additionally, extracts
from Boswellia species gum resins might
possess anti-cancer activities, based on their
anti-proliferative and pro-apoptotic activities
in rat astrocytoma cell lines and Clinically,
extract from the resin reduces the
peritumoral edema in glioblastoma
patients (Winking, 2000) and in human
leukemia cell lines (Hostanska, 2002), as
well as their anti-carcinogenic activity in
chemically induced mouse skin cancer
models (Huang, 2000). The pharmacological
characteristics and clinical efficacy
of Boswellia serrata have been studied, with
research published and systematically
reviewed in the medical literature (Ernst,
2008). These results suggest that
frankincense resin contains active
ingredients that modulate important
biological activities. B. serrata flowers and
leaves showed significant antibacterial
activity (Mohammed Aman et al., 2010). In
addition B. serrata has versatile
pharmacological activities (Arshiya Sultana
et al., 2013).
However, there are no enough scientific
reports to support these supposed
antimicrobial activity. The present
investigation was undertaken which deals
with the evaluation of antimicrobial activity
of frankincense resin extract of Boswellia
serrata.
Materials and Methods
The crude gum is collected and processed.
The collected material is dried under shade
and made into powder and subjected to hot
percolation by using soxhlet apparatus with
water. The extract was filtered using
Whatsman-No. 1 filter paper and the
extraction procedure were repeated three
times. The filtrate was used for the
biological assay.
Microorganisms
Clinical isolates of Gram positive (Bacillus
subtilis, Staphylococcus aureus and
Streptococcus pneumonia) and Gram
negative (Escherichia coli, Klebsiella
pneumonia, Pseudomonas aeruginosa,
Enterobacter aerogenes and Proteus
vulgaris,) authentic bacterial strains were
used in the study which are procured from
Institute of Microbial Technology
(IMTECH), Chandigarh. The stock culture
are maintained at Division of Animal
Biotechnology, Sri Venkateswara
University. All the microorganisms were
maintained at 4°C on nutrient agar slants.
Preparation of the Bacterial Suspension
The turbidity of each of the bacterial
suspension was prepared to match to a 0.5
McFarland standard (1.5x108 CFU/ml).
Measure the turbidity with the aid of a
spectrophotometer at an optical density
0.08-0.13 and turbid suspension at 625 nm
as per Bauer-Kirby Method (1966).
Determination of antimicrobial activity
Culture of the bacterial organism was
aseptically introduced and evenly spread
using sterile ‘L’ rod on the surface of sterile
Mueller Hinton agar (M173/M1084,
HiMedia) plates. The agar disc diffusion test
is the most convenient and widely used
method for routine antimicrobial
susceptibility testing according to CLSI
(Clinical Laboratory Standards Institute;
formerly NCCLS). 25 µL of different
concentrations (25, 50,75,100 mg/ml) of the
resin coarse powder extract were added on
Whatman No. 1 filter paper disc (6mm) and
was inoculated with a loopful of the test
organism previously diluted to 0.5
McFarland turbidity standards seeded on the
medium. The experiment was run in

Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 1095-1101
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triplicate for each extract and each organism
tested. The plates were incubated at 37°C
for 24 hours and the zones of inhibition were
measured in millimeters using a calibrated
instrument like zone scale (HiMedia).
DMSO was used as a negative control.
These inoculated tubes were then incubated
Control experiments comprising inoculums
without the extract were set up.
Determination of MIC
The determination of the MIC was done
with different dose levels of (25, 50, 75
and100 mg/ml) of extract. The procedure
was repeated on all the test organisms using
the standard antibiotic Ciprofloxacin (5
µg/mL Disc, Himedia, Mumbai, India).
The statistical analysis was undertaken using
t-test in SPSS statistics software (Version
20, IBM Corporation, New York, USA),
considered significant when p≤0.05.
Result and Discussion
In this study the resin extract of
commercially available Boswellia serrata
powder was evaluated for their antimicrobial
activity. Preliminary antimicrobial screening
assay of commercially available Boswellia
serrata gave relatively wide inhibition zone
against the test strains. Minimum inhibitory
concentration (MIC) is defined as the lowest
concentration that will inhibit the growth of
a test organism over a defined interval
related to the organism’s growth rate, most
commonly 18-24h (Turnidge et al., 2003).
The zone size is inversely proportional to
the minimum inhibitory concentration
(MIC). The MIC values revealed different
activities against the eight tested bacteria at
the concentrations 10, 25, 50, 100mg/ml
(Table-1). All the bacterial strains were
compared with Ciprofloxacin under the
same experimental conditions.
The highest antimicrobial activity was
observed on E. coli with zone of inhibition
as 21.87±0.98 and the lowest effect was on
E. aerogenes with a zone of inhibition of
11.67±1.00 mm. Whereas, Standard showed
highest activity against E.aerogenes
(30.27±1) and lowest activity against S.
pneumoniae (20.07±0.74). The order of
inhibitory activity of extract against
different organisms is: E. coli> S. aureus>
B. subtilis> S. typhi> K. pneumoniae> S.
pneumoniae> E. aerogenes> P. vulgaris.
The order of inhibitory activity of standard
against different organisms are summarized
as P. vulgaris> E.coli> S. aureus> E.
aerogenes> P. aureginosa> B. subtilis> K.
pneumonia (Graph-1). This shows that the
studied Boswellia serrata frankincense
extract mostly exhibited bacteriostatic
effects.
Extract inhibitory activity was statistically
compared to the inhibitory activity of
standard and observed that extract inhibitory
activity on the microbes has significantly
lower activity than the standard (P≤0.01,
P≤0.001).
Disc diffusion test is a qualitative test
method. The recommended medium for disc
diffusion testing is Mueller-Hinton agar
(MH; Himedia, Mumbai, India) (CLSI,
2012). This medium demonstrates good
batch-to-batch reproducibility, and supports
the growth of most non fastidious bacterial
pathogens (Jorgensen and Turnidge, 2003).
Well-variant of the diffusion method was
more sensitive and best conditions for the
determination of minimal inhibitory
concentration (Valgas et al., 2007).
Dimethyl sulfoxide (DMSO) used as solvent
for natural as well as synthetic antibacterial
compounds (Wadhwani et al., 2009;
Houghton and Raman, 1998).

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Graph.1 Antimicrobial efficacy of Boswellia serrata frankincense resin
extract against selected bacterial pathogens
Values are the Mean ± SD; Astringent indicate level of significance (**=p≤0.01, ***=p≤0.001)
Table.1 Minimum Inhibitory Concentrations (MIC)
S. No
Test Organism
G+ / G-
I
II
III
IV
1.
Bacillus subtilis
G+
++
+
+
*
2.
Staphylococcus aureus
G+
+
+
*
-
3.
Streptococcus pneumoniae
G+
+
+
+
*
4.
Escherichia coli
G-
+
*
-
-
5.
Klebsiella pneumoniae
G-
++
+
+
*
6.
Pseudomonas aeruginosa
G-
++
++
+
+
7.
Enterobacter aerogenes
G-
++
++
+
*
8.
Proteus vulgaris
G-
++
++
++
+
I = 25mg/ml; II =50 mg/ml; III =75 mg/ml; IV =100 mg /ml,*=MIC, - = No growth, + =
Moderate growth, ++ =Dense growth, G+ = Gram Positive bacteria, G- = Gram Negative bacteria,

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However, exclusive focus on individual
biochemical targets neglects the fact that
strong synergy of multiple constituents in a
crude drug may prove more potent and
effective than any single purified compound,
or that interactions of co-occurring
phytochemicals may help nullify the toxic
effects of individual constituents. So while it
is important to understand the active agents
within medicinal plants, it should also be
with caution that we extract and use
constituents in isolation. In this study the
Boswellia extract have shown greater
antimicrobial activity which may explain
anonymous claim on the topical use of
Boswellia serrata frankincense for microbial
infections.
In Conclusion, the screening of
antimicrobial activity performed on resin
fractions of 25, 50, 75, 100 mg/ml of
Boswellia serrata, traditionally important
medicinal plant proved to be a bacteriostatic
agent. The current work will provide new
reference data for the drug development and
possesses the ability to inhibit pathogenic
bacteria. Further studies should be done on
fractionation and identification of bioactive
constituents which are responsible for
antibacterial activity.
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