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National Journal of Health Sciences, 2017, 2, 61-66 61
© 2017 NiBD Publicatons www.njhsciences.com
Research Article
Potential of Red Sandalwood (Adenanthera pavonina L.) as an Antibacte-
rial Agent against Clinical Isolates
Tanveer Abbas1,*, Faryal Liaquat1, Saira Yahya2 Farhana Tasleem3, Iqbal Azhar3 and Zafar Alam
Mehmood4
1FSRG, Department of Microbiology, University of Karachi, 75270, Karachi, Pakistan.
2Department Of Microbiology,University Of Karachi, Pakistan.
3Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Pakistan.
4Colorcon Limited, Crossways, Victoryway, Kent, Dartford, England.
Abstract: The emergence of resistant pathogens is a leading cause of morbidity worldwide. For the treatment of diseases caused by these
resistant pathogens, the use of medicinal plants as an alternative to synthetic drugs is increasing. Therefore, the aim of this study was to eval-
uate the efficacy of hexane and ethanolic extracts of a natural plant Adenanthera pavonina L. Antibacterial activity of Adenanthera pavonina
L. leave extracts and pure compound (β-sitosterol glucoside) against Enterrococcus spp., S. aureus, P. aeruginosa, S. typhi, E. coli, Proteus
spp., K. pneumonia and A. baumanii, was carried out by well-diffusion assay and micro-dilution technique. Moreover, time-kill tests were
carried out to assess the antimicrobial activity of the Adenanthera pavonina L. extracts against E. coli. The results showed good antibacterial
potential of Adenanthera pavonina L. extracts against the clinical isolates tested.
Keywords: Antibacterial potential, β-sitosterol glucoside, Adenanthera pavonina L.
doi.org/10.21089/njhs.22.0061
INTRODUCTION
Antibiotics, which are either derived from microorganisms
or synthesized chemically, provide the base of clinical thera-
py. Every year, two to three antibiotics were sprung into the
global market, but due to the emergence of multi-drug re-
sistant microbial strains, scientist are looking forward for the
development of alternative and novel drugs [1]. Natural
sources such as plants, offer a wide variety of natural medic-
inal compounds for therapeutic treatments [1, 2]. Their use
for the treatment of diseases is increasing day by day be-
cause of their added advantages such as low toxicity, good
therapeutic performance, cost-effectiveness and eco-
friendliness [3-6]. In order to understand the antimicrobial
efficacy of these medicinal plants, their properties should be
thoroughly investigated.
The most common plants used for therapeutic purposes
worldwide are Lannea kerstingii, Curcuma xanthorrhiza,
Senna alata, Moringa oleifera, Adenanthera pavonina L. and
Pangium edule. The plant Adenanthera pavonina L., native
to the subcontinent was chosen in this study. Adenanthera
*Address correspondence to this author at the Department of Microbiology,
University of Karachi, 75270, Karachi, Pakistan.
E-mail: taabbas@uok.edu.pk
pavonina L. is also known as Red Bead Tree (Red sandal
wood) and belongs to the family Fabaceae [7]. Various parts
of this plant have been traditionally used for treating diar-
rhea, gout and inflammations [8]. Its bark is typically used
for the treatment of gonorrhea, haematuria, ulcers and it is
also used as an ointment [9-16].
Several documented phytochemical studies on this plant re-
vealed the presence of secondary metabolites, mainly flavo-
noids, triterpenoids, tannins, saponins and sterols [5, 10, 11].
The chief constituents are flavonoid compounds [12, 13]. It
is used as an aseptic paste and also used to treat boils and
inflammation [12, 14]. Its wood is red in color and extremely
hard wood timber is used for building purposes and furniture
making [12]. It is also able to fix nitrogen [5, 15].
Plants generally synthesize ethyl sterols (sitosterols) while
fungi, algae and protozoa, synthesize methyl sterols
(ergosterols) [17]. Among the phytosterols, predominant is
β-sitosterol which are structurally similar to cholesterol. β-
sitosterols are present in natural foods and are considered as
the health promoting constituents. They also possess phar-
macological activities such as anti-inflammatory activity,
immunomodulatory activity, chemoprotective effects, induc-
ing apoptosis and angiogenic effect [18].
62 National Journal of Health Sciences, 2017, Vol. 2, No. 2 Tanveer Abbas
Owing to the therapeutic properties of Adenanthera
pavonina L., this study was carried out to evaluate the anti-
bacterial potential of leave extracts and a pure compound- β-
sitosterol glucoside, isolated from Adenanthera pavonina L.
MATERIALS AND METHODS
Microbial Strains
Total 8 clinical isolates were used in this study which were
provided by a clinical laboratory. Out of these 8 isolates, two
were Gram-positive (Enterrococcus spp., S. aureus) and six
were Gram-negative (P. aeruginosa, S. typhi, E. coli, Pro-
teus spp., K. pneumonia and A. baumanii).
Preparation of 0.5 McFarland
McFarland standards are used as a reference to adjust the
turbidity of bacterial suspensions. It was prepared by mixing
0.05 ml of 1% BaCl2.2H2O with 9.95 ml of 1% H2SO4. A600
was taken by using a spectrophotometer and stored in a re-
frigerator until use [19].
Preparation of Inoculum
Pure microbial isolates were streaked onto nutrient agar me-
dium. Subsequently, isolated colonies were transferred in
sterile PBS and turbidity of the suspension was adjusted to
0.5 McFarland standard [20].
Preparation of Extracts
Leaves of Adenanthera pavonina L. were soaked in hexane
and ethanol for fifteen days, filtered and filtrate was evapo-
rated under reduced pressure by means of rotary evaporator,
while the pure compound was isolated from the leaves of
Adenanthera pavonina L. [21].
Determination of Antimicrobial Potential
The antimicrobial activity of the extracts and pure compound
i.e. β-sitosterol glucoside was evaluated against the clinical
isolates. The procedures, well-diffusion assay and minimal
inhibitory concentration (MIC) were carried out as per Clini-
cal Laboratories Standards Institute (CLSI) guidelines to
check the antibacterial sensitivity [20, 21]. Time-kill assay of
the extracts and compounds against E. coli was also investi-
gated. Berberis vulgaris extract was used as a positive con-
trol throughout the study.
Well-Diffusion Assay
Sensitivity test was performed using agar well-diffusion
method. A lawn of bacterial inoculum (matched with 0.5
McFarland turbidity standard) was made on Mueller-Hinton
agar plate. The plant extract or β- sitosterol glucoside (100
μl) was added in a 7 mm well punched aseptically in the agar
plate with a sterile cork borer and was incubated at 37°C for
24 h. The sensitivity was determined based on the diameter
of the zone of inhibition around the well [20-24].
Minimum Inhibitory Concentration (MIC)
The sensitivity of microbes to ethanolic extract was meas-
ured by micro dilution method using 96-well microtiter
plate. The extract was serially diluted (two-fold) in Mueller-
Hinton broth, 10 μl culture was added in each well along
with the positive and negative controls and the plates were
incubated at 37°C for 24 h. A540 was read by ELISA plate
reader (Tecan sunrise, Switzerland). MIC’s were calculated
in percentages and in mg/ml.
Time-Kill Assay
To determine at which time of bacterial growth the test com-
pound exerted its killing effect, a time kill assay was carried
out. The experiment was performed by inoculating test or-
ganism (E. coli) in Nutrient broth and incubating at 37°C for
24 h. It was matched with 0.5 McFarland and broth culture
was diluted to 10-3 dilution. 1 ml of culture was added with
0.1 ml of extract in dry cuvettes. A600 was measured after
different time interval at 0 min, 30 min, 60 min, 120 min,
240 min and 1440 min.
Statistical Analysis
Data was organized and tabulated using Microsoft word and
Excel 2007. Pearson’s correlation was calculated for all three
extracts vs. natural antimicrobial agent Berberis vulgaris.
Time-kill assay graphs were made by using online DMFit,
web edition (Dynamic modeling fit).
RESULTS AND DISCUSSIONS
Many infectious diseases have been known from history to
be treated with herbal remedies due to their antibacterial
activity against a large number of microbes as well as their
reasonable cost [25-29]. The activity of plant extracts against
bacteria is due to the presence of phytoconstituents, which
may vary due to certain factors such as locality of plant
growth, harvesting and extraction methods.
In this study two methods were used to determine the effica-
cy of Adenanthera pavonina L. against the bacterial strains
i.e. the well-diffusion method and the microdilution method
(Minimum Inhibitory Concentration).
Antibacterial Activity Determination by Well-Diffusion
Assay
The results indicate that the Adenanthera pavonina L. leave
extracts showed antibacterial activity against the respective
clinical isolates with different diameter of zone of inhibition
and only Proteus spp. showed resistance to all extracts (Ta-
ble 1). The ethanolic extract was effective against Entero-
coccus spp., E. coli, S. aureus and A. baumannii with zone of
inhibitions of 7 mm, 17.5 mm, 8 mm and 17 mm respective-
Potential of Red Sandalwood (Adenanthera pavonina L.) National Journal of Health Sciences, 2017, Vol. 2, No. 2 63
ly. S. aureus, P. aeruginosa and S. typhi showed sensitivity
with zones of inhibition of 10 mm and Enterococcus spp.
with 11 mm diameter to hexane extract of Adenanthera
pavonina L. Similar results have been reported by several
workers [30, 31].
The antibacterial activity showed by Adenanthera pavonina
L. pure compound (β-sitosterol glucoside) to S. aureus, E.
coli and A. baumanii measured in diameter was 15 mm, 9
mm and 11 mm respectively. The potential of β-sitosterol as
an antibacterial agent from Senecio lyratus is documented by
Kiprono et al. in 2000 [32]. In a study by Sen et al. (2012),
the antimicrobial activity of pure β-sitosterol is reported
ranging from 10 to 14 mm for P. aeruginosa, E. coli, K.
pneumonia, and S. aureus which is nearly equal to the stand-
ard Gentamicin by well-diffusion method.
Table 1. Zone of inhibition. (-) indicates no inhibition Berberis vulgaris as a positive control, correlations were significant for hexane (r =
0.808, r2 = 0.654) and pure compound (β-sitosterol glucoside) (r = 0.252, r2 = 0.0063) and insignificant for ethanolic extract (r = -0.084, r2 =
0.0071).
Clinical Isolates
Pure Compound
(mm)
Ethanolic Extract
(mm)
Hexane Extract
(mm)
Berberis Vulgaris (Positive Control)
(mm)
S. aureus
15
8
10.5
21
Enterococcus
-
7
11
16
P. aeruginosa
-
-
10
19
E. coli
9
17.5
-
15
S. typhi
-
-
10
15
A. baumanii
11
17
-
10
K. pneumonae
-
-
-
11
Proteus spp
-
-
-
11
Table 2. Minimum Inhibitory Concentration presented in percentages and in mg/ml, significant correlation for hexane (r = 0.0922,
r2 = 0.0085) and pure compound (r = 0.0315, r2 = 0.001) vs. positive control (Berberis vulgaris) while insignificant for ethanolic extract
(r = -0.674, r2 = 0.4553).
Clinical Isolates
Ethanolic
Extract
Hexane Extract
(mg/ml)
Pure Compound
(mg/ml)
Berberis Vulgaris (Positive Control)
S. aureus
5%
25
25
25%
P. aeruginosa
10%
12.5
25
6.25%
S. typhi
10%
25
50
6.25%
E. coli
10%
25
25
3.1%
Enterococcus
10%
25
25
6.25%
Proteus
0.625%
25
25
12.5%
K. pneumoniae
10%
25
25
1.5%
A. baumanii
10%
25
12.5
3.1%
64 National Journal of Health Sciences, 2017, Vol. 2, No. 2 Tanveer Abbas
Fig. (1).Time kill of Berberis vulgaris positive control fitted in
DmFit (Dynamic modeling fit) r2 = 0.997 and SE of fit 0.00748.
Fig. (2).Time kill of hexane extract of Adenanthera pavonina fitted
in DmFit (Dynamic modeling fit) r2 = 0.947 and SE of fit 0.0638.
Antibacterial Activity Determination by Minimum inhib-
itory Concentration (MIC)
In clinical laboratories, the micro-dilution method is used
routinely as a quantitative reference method therefore it was
used in this study. The minimum inhibitory concentration
calculated for Adenanthera pavonina L. pure compound (β-
sitosterol glucoside) was 12.5 mg/ml to 50 mg/ml (Fig. 2),
whereas the MIC of the hexane and ethanolic extracts ranged
from 12.5 mg/ml to 25 mg/ml and 0.625% to 10% respec-
tively. Some clinical isolates failed to show results in well-
diffusion assay while minimum inhibitory concentration was
determined against them which might be due to reasons like;
heavy bacterial growth, large inoculum size or the insolubili-
ty of extracts in agar.
Time-Kill Assay
Time-kill assay of hexane and ethanolic extracts as well as of
β-sitosterol glucosidase was calculated against E. coli. The
results were compared with the positive control Berberis
vulgaris (Fig. 1). Time kill of hexane extract of Adenanthera
pavonina L. showed the bactericidal activity till two hours
after which the extract lost its activity (Fig. 2). Ethanolic
extract showed decrease in bacterial count till one hour and
stationary phase was observed after that as shown in (Fig. 3).
The pure compound β-sitosterol glucoside also exerted its
killing effect and a decrease in bacterial count was observed
as shown in (Fig. 4). The results demonstrated that the test
compounds exerted their killing effect by acting on the
growing cells.
Fig. (3).Time kill of ethanolic extract of Adenanthera pavonina
fitted in DmFit (Dynamic modeling fit) r2 = 0.947 and SE of fit
0.0638.
Fig. (4). Time kill of pure compound β-sitosterol glucoside of
Adenanthera pavonina L. fitted in DmFit (Dynamic modeling fit) r2
= 0.701 and SE of fit 0.208.
CONCLUSION
This study demonstrated that Adenanthera pavonina L. ex-
tracts or their bioactive components, if found non-toxic in
animal studies and clinical trials, can be effectively used
against the most common bacterial infections.
Potential of Red Sandalwood (Adenanthera pavonina L.) National Journal of Health Sciences, 2017, Vol. 2, No. 2 65
CONFLICT OF INTEREST
Declared none.
ACKNOWLEDGEMENT
The authors are thankful to Dean Faculty of Science, Univer-
sity of Karachi, Karachi. 75270, Pakistan, for providing the
financial support for the research.
REFERENCES
[1] Jayamani E, Rajamuthiah R, Larkins-Ford J, Fuchs B, Conery A,
Vilcinskas A, et al. Insect-derived cecropins display activity against
acinetobacterbaumannii in a whole-Animal high-throughput
caenorhabditiselegans model. Antimicrob. Agents Chemother.,
2015; 59(3): 1728-1737. DOI: 10.1128/aac.04198-14
[2] Ocheng F, Bwanga F, Joloba M, Borg-Karlson K, Gustafsson A,
Obua C. Antibacterial activities of extracts from Ugandan
medicinal plants used for oral care. J. Ethnopharmacol., 2014;
155(1): 852-855. DOI: 10.1016/j.jep.2014.06.027
[3] Yakubu T., SunmonuO, Lewu B, Ashafa O, Olorunniji J,
EddouksM. Efficacy and safety of medicinal plants used in the
management of diabetes mellitus. J. Evid. Based Complementary
Altern. Med., 2014; 1-2. DOI: 10.1155/2014/793035
[4] Vargas N, Ceolin, T, Souza A, Costa M, Ceolin S, Heck R.
Medicinal plants used in the process of wound healing by growers
in the south region of the RS state. R. Pesq. Cuid. Fundam. Online.
2014; 6(2); 550-560. DOI: 10.9789/2175-5361.2014v6n2p550
[5] Pagano MC, Dhar PP. Fungal pigments: an overview. Fungal bio-
molecules: sources, applications and recent developments. USA:
Wiley-Blackwell; 2015. 173. DOI: 10.1002/9781118958308
[6] Friendly E, Ideas B. Eco friendly business info. Solar Energy.
2014.
[7] Watal G, Dhar P, Srivastava S, Sharma B. Herbal medicine as an
alternative medicine for treating diabetes: The global burden. evid.
based complement. Alternat. Med., 2014: 1-2.
DOI: 10.1155/2014/596071
[8] Tiwari R, Chakrabort S, Saminathan M, Kumar A, Karthik K, Wani
M, et al. Evidence based antibacterial potentials of medicinal plants
and herbs countering bacterial pathogens especially in the era of
emerging drug resistance: an integrated update. Int. J. Pharmacol.,
2014; 10(1): 1-43. DOI: 10.3923/ijp.2014.1.43
[9] Jawallapersand P, Mashele SS, Kovačič L, Stojan J, Komel R,
Pakala SB, et al. Cytochrome P450 monooxygenase CYP53 family
in fungi: comparative structural and evolutionary analysis and its
role as a common alternative anti-fungal drug target. Plo. S. one.
2014; 9(9): e107209. DOI: 10.1371/journal.pone.0107209.
[10] Tan R., Saga seed tree. 2017. Retrieved from:
http://www.naturia.per.sg/buloh/plants/saga_tree.htm
[11] Adedapo AD, Osude YO, Adedapo AA, Moody JO, Adeagbo AS,
Olajide AO, et al. Blood pressure lowering effect of Adenanthera
pavonina seed extract on normotensive rats. Net. Prod. Res., 2009;
3(2): 82-9.
[12] Adedapo A, Olayinka J, Abiodun O, Oyagbemi A, Azeez O,
Adedapo A, et al. Evaluation of antimalarial and antioxidant
activities of the methanol seed extract of Adenanthera pavonina
(Linn) in Plasmodium berghei infected mice. Asian J. Med. Sci.,
2014; 5(4).
[13] Sumathy R, Vijayalakshmi. M, Deecaraman. M, Sankaranarayanan
S, Bama P, Ramachandran J. Screening of secondary metabolites,
antioxidant and antimicrobial activity from the petals of
moringaoleifera. World J. Pharm. Sci., 2014; 3(6): 1829-1843.
[14] Saxena, H, Soni, A, Mohammad N, Choubey S. Phytochemical
screening and elemental analysis in different plant parts of Uraria
picta Desv.: A Dashmul species. J. Chem. Pharm. Res., 2014; 6(5),
756-760.
[15] Mukda S, Subhadhirasakul S. Herbal medicines and hemorrhoid
disease. J. Thai Traditional Alternative Med., 2014; 12(2): 122-132.
[16] Mohammed S., Abou Zeid H, El-Kashoury A, Sleem A, Waly A. A
new flavonol glycoside and biological activities of Adenanthera-
pavonina L. leaves. Nat. Prod. Res, 2014; 28(5): 282-289. DOI:
0.1080/14786419.2013.856903
[17] Silva IK, Soysa P. Evaluation of phytochemical composition and
antioxidant capacity of a decoction containing Adenanthera
pavonina L. and Thespesiapopulnea L. Pharmacogn. Mag., 2011;
7(27): 193-199.
[18] Richard Van C, Winrock international NFT highlights. A quick
guide to useful nitrogen fixing trees from around the world: detailed
fact sheet on habit, habitat, distribution, uses. 1993, NFTA 96-01.
[19] Vasavi HS, Arun AB, Rekha PD. Anti-quorum sensing potential of
Adenanthera pavonina. Pharmacognosy Res., 2015; 7(1): 105-109.
[20] Performance standards for antimicrobial disk and dilution
susceptibility tests for bacteria isolated from animals; approved
standard-3rd edition CLSI Document M31-A3; 2008; 28: 65.
[21] Jorgensen JH, Hindler JA, Barnar K, Citron DM, Cockerinn FR,
Fritsche TR, et al. Methods for antimicrobial dilution and disk
susceptibility testing of infrequently isolated or fastidious bacteria.
Clinical and laboratory standards institute. Approved guideline-2nd
edition, CLSI Document M45-A2, 2010; 30: 18.
[22] Shrafee T, Rahman M, Chakraborty A, Prodhan H. Antibacterial
potentiality of red sandalwood callus against pathogenic isolates of
aeromonas and pseudomonas. Universal J. Plant Science. 2014;
2(4): 86-91. DOI: 10.13189/ujps.2014.020402
[23] Shigemura K, Takase R, Osawa K, Takaba K, Nomi M, Fujisawa
M,ArakawaS. Emergence and prevention measures for multidrug
resistant Pseudomonas aeruginosa in catheter-associated urinary
tract infection in spinal cord injury patients. Spinal Cord. 2015;
53(1): 70-74. DOI: 10.1038/sc.2014.154
[24] Aghamohammadi M, Faraji M, Shahdousti P, Kalhor H, Saleh A.
Trace determination of lead, chromium and cadmium in herbal
medicines using utrasound assisted emulsification micro-extraction
combined with graphite furnace atomic absorption spectrometry.
Phytochem. Anal., 2015; 26(3): 209-214. DOI: 10.1002/pca.2554
[25] Shalaby S, Ismail M, Mansour N, Abbas H, Gupta N. Patterns of
herbal use among patients attending family practice centers. El.
Med. J., 2015; 2(4): 366. DOI: 10.18035/emj.v2i4.348
[26] Laslett L, Jin X, Jones G. Efficacy and safety of plant-derived
products for the treatment of osteoarthritis. Botanics. 2015; 5(5): 1-
20. DOI: https://doi.org/10.2147/BTAT.S33431
[27] Murthy N, Georgiev I, Park Y, Dandin S, Paek Y. The safety
assessment of food ingredients derived from plant cell, tissue and
organ cultures: a review. Food Chem., 2015; 176: 426-432. DOI:
10.1016/j.foodchem.2014.12.075
[28] Adnan M., Bibi R, Mussarat S., Tariq A, Shinwari ZK.
Ethnomedicinal and phytochemical review of Pakistani medicinal
plants used as antibacterial agents against E. coli. Ann. Clin.
Microb. Anti. J., 2014; 13(1): 40. DOI: 10.1186/s12941-014-0040-6
[29] Nwankwo IU, Ukaegbu-Obi KM, Mushtaq A, Pervez S, Hussain S,
Asif M, et al. Preliminary phytochemical screening and
antibacterial activity of two Nigerian medicinal plants (Ficus
66 National Journal of Health Sciences, 2017, Vol. 2, No. 2 Tanveer Abbas
asperifolia and Terminalis catappa), J. Med. Plant Herb. Ther.
Res., 2014.
[30] Chemat F. Vian MA, Cravotto G. Green extraction of natural
products: concept and principles. Int. J. Mol. Sci., 2012; 13(7):
8615-8627. DOI: 10.3390/ijms13078615
[31] Sen A, Dhavan P, Kshitiz K, Singh S, Tejovathi G. Analysis of IR,
NMR and antimicrobial activity of β-Sitosterol isolated from
Momordica charantia. Sci. Secure. J. Biotech., 2012; 1(1): 9-13.
Received: June 13, 2016 Revised: March 27, 2017 Accepted: April 03, 2017
© 2017 National journal of health sciences.
This is an open-access article.
