This document summarizes a study that evaluated the antimicrobial activity of extracts from Terminalia arjuna bark. Methanol and water extracts were tested against bacterial and fungal clinical isolates. The methanol extract showed higher total phenolic and flavonoid content. Both extracts inhibited the growth of pathogenic bacteria like S. aureus and fungi like Candida species. Minimum inhibitory concentration values ranged from 0.04-1 mg/mL. Comet assay results also indicated that the extracts induced DNA damage in Candida tropicalis. Overall, the study demonstrated the antibacterial and antifungal properties of T. arjuna bark extracts.

1. Clinical isolates of urinary tract
infection and candidiasis inhibited by
T. arjuna bark extract
Dr. Diganta Dey
Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata- 700068
Department of Pharmaceutical Technology, Jadavpur University, Kolkata- 700032

2. INTRODUCTION
• 25% of the total 57 million annual deaths that occur
worldwide are caused by pathogenic microorganisms
(The Lancet Infect Dis, 2009; 9:365-375).
• In 1985, WHO estimated that 80% of the people
living in developing countries were dependent on
traditional herbal health-care, while about 25% of all
prescriptions dispensed from community pharmacies
in the USA contained either plant extracts, or active
principles prepared from higher plants (Bull WHO,
1985;63:965-981).
• Terminalia arjuna, Wight & Arn. (family Combretaceae), is traditionally
renowned as a cardiac stimulant, and also recommended by many indigenous
systems of medicine to address miscellaneous problems like urinary
discharges, anaemia, bilious affliction, and as an antidote to poison (The
Indian Materia Medica, Vol. 1., 3rd edn. 1198-1202).
• However, its antimicrobial profile did not receive adequate attention until
recently, and there is ample scope to undertake further investigation in this
regard.

3. MATERIALS AND METHODS
• Plant material. Stem-bark of Terminalia arjuna Wight & Arn.
collected fresh from Baruipur, South 24 Parganas, West Bengal, was
duly authenticated at Botanical Survey of India, Shibpur, Howrah.
• Preparation of extracts. The plant material was shade dried and
ground to powder. Three portions were chloroform, methanol and
water, and refluxed to obtain the respective extracts. After filtration,
the non-polar solvents were removed in a rotary evaporator. The water
extract concentrated and freeze-dried in a lyophiliser. The yield (%
w/w) of the crude extracts obtained from chloroform, methanol and
water were recorded and named as TAC, TAM and TAW,
respectively.
• Phytochemical analysis. The secondary metabolites present in the
extract were detected qualitatively by the characteristic colour changes
occurring upon treatment with specific reagents, e.g., alkaloids with
Dragendorff reagent, tannins with ferric-chloride solution, flavonoids
with sodium hydroxide solution, triterpenoids with acetic anhydride in
ethanolic solution, and reducing sugars with Benedict’s reagent.
Saponins could be detected by the ability to produce stable foam upon
intermittent shaking of an aqueous suspension of the extract.

4. MATERIALS AND METHODS
• Total phenolic content. A stock solution 0.02 mL (10 mg/mL) + 1.58
mL of distilled water + 0.1 mL of Folin Ciocalteau reagent (0.2 N) +
Na2CO3 solution (7.5%; 0.3 mL). 30 min incubation in dark.
Absorbance at 765 nm was measured in a UV-spectrophotometer. A
standard curve was prepared using different concentrations of gallic
acid and the total phenolic content was expressed as gallic acid
equivalents (GAE; mg/g of sample).
• Total flavonoid content. A stock solution of 0.1 mL (10 mg/mL) +
0.3 mL of distilled water containing NaNO2 (5%; 0.03 mL) + AlCl3
(10%; 0.03 mL) was added after 5 min + After another 5 min, the
reaction mixture was treated with 0.2 mL of NaOH (1 mM). Finally,
the volume was made up to 1 mL with distilled water and the
absorbance measured at 415 nm. Quercetin dihydrate (SRL; Mumbai,
India) was used to prepare the calibration graph. The test was carried
out thrice, and the results were expressed as quercetin equivalent (QE;
mg/g of sample).

5. MATERIALS AND METHODS
• Microorganisms for study. A total of thirty pathogenic microbial
cultures including nine ATCC strains of bacterial and fungal origin
were taken for this study. Fifteen of the bacteria and six fungal strains
were isolated from clinical specimen obtained from patient samples,
and identified by standard laboratory protocol.
• Antimicrobial activity. The plant extracts were dissolved in DMSO at
a concentration of 10 mg/mL, and tested for antimicrobial activity by
the agar well diffusion assay. MHB/SDB was adjusted to the final
inoculum density of 1x107cfu/mL (by 0.5 McFarland standard) on
molten MHA/SDA plates. After solidification, wells (diameter 9 mm)
were made with a sterile borer in the inoculated MHA/SDA plates.
100µL solution containing 1 mg of each extract was dispensed in the
wells, while DMSO was also tested as the vehicle control. Penicillin G,
Streptomycin, Gentamicin and Amphotericin-B used as positive
controls in this assay. Antimicrobial activity was expressed as the
diameters of inhibition zones produced around each well by the plant
extracts and antibiotics, and was measured after 24 h of incubation at
37°C.

6. MATERIALS AND METHODS
• Determination of minimum inhibitory concentration (MIC). The
MIC of the plant extracts against the tested bacteria was determined by
broth micro-dilution procedure. Stock solutions (10.24 mg/mL) of TAM
and TAW were prepared in DMSO, and serially diluted in MHB/SDB at
concentrations of 5.12, 2.56, 1.28, 0.64, 0.32, 0.16, 0.08 and 0.04 mg/mL
in a 96-well microtitre plate. The broth culture containing 0.5 McFarland
(1x108 cfu/mL) inoculum density was then introduced to each of the
microtitre wells at 1:10 ratio to maintain final inoculum density of
1x107cfu/mL. Microtitre plates were incubated for 18 h at 37ºC (35ºC of
fungal), and the presence of visible growth in each well was inferred by
measuring OD at 630 nm using ELISA reader.
• Determination of drug resistance profile of the isolated bacteria.
Antibacterial susceptibility studies were carried out by CLSI
recommended Kirby and Bauer disk diffusion technique using
commercially available antibiotic disks. Bacterial culture in peptone
water containing 0.5 McFarland turbidity (1x108 cfu/mL) was swabbed
in MHA plate. Antibiotic disks were placed on it by maintaining about
20 mm distance with each other. Inhibition zone diameter was measured
after overnight incubation at 37ºC and results were interpreted as per
CLSI guidelines.

7. MATERIALS AND METHODS
• Evaluation of T. arjuna extracts induced DNA damage in Candida tropicalis by alkaline
single cell gel electrophoresis (SCGE, or Comet assay) Candida tropicalis ATCC 750 cell
suspension at 0.5 McFarland (~ 1 x 108 Cells/ml) was diluted in nuclease free water (to
prepare negative control), 100 µM H2O2 solution (to prepare positive control), TAW and
TAM (1mg/ml) solution at a ratio of 1:10 (v/v) and kept for 30 minutes at 4ºC. After that,
aliquots from negative control, positive control, TAW and TAM treated samples were
further diluted in phosphate buffered saline (PBS; Ca2+ and Mg2+ free; pH 7.4) at a ratio
of 1:100 (v/v) to attain final cell density 1 x 105/ ml. SCGE was performed using comet
assay reagent kit (Trivigen, Gaithersburg, MD, catalog # 4250-050-K) according to the
manufacturer’s instructions. Briefly, C. tropicalis cell suspension (1 x 105 cells/ml) in PBS
was combined with 1% molten low melting agarose at 37°C at a ratio of 1:10 (v/v) and
transferred to comet slide. After gelling time (~ 30 minutes), the slide was immersed
respectively in pre-chilled (4ºC) lysis solution and alkaline solution (200 mM NaOH, 1mM
EDTA, pH > 13) for 45 minutes in each. Then electrophoresis of slide was conducted in
pre-chilled (4ºC) alkaline electrophoresis solution (200 mM NaOH, 1mM EDTA, pH >
13) for 30 minutes at 25 volt and 300 mA (1.5 V/cm), and then washed in deionized water,
dehydrated in 70% ethanol and stained with SYBR® Green I (maximum excitation /
emission was 494 nm/ 521 nm) diluted in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM
EDTA). Fluorescent comet patterns were examined with a MOTIC BA400 microscope
under 200× magnification and fluoroisothiocyanate (FITC) filter combination and images
captured by a charge-couple device (CCD) camera.
• Comet Score Freeware v1.5 (TriTek Corporation) was used to measure 15 comet
parameters.

8. RESULT
alkaloids tannins flavonoids triterpe
noids
saponi
ns
reducin
g
sugars
phenolic
contents
(mg
GAE/g)
flavonoid
contents
(mg QE/g)
TAM + + + + + + 289.2 ± 6.9 18.0 ± 0.0
TAW + + + + + + 256.2 ± 3.1 14.4 ± 0.2
TAC + - - + - + N.D. N.D.
Phytochemical analysis:

9. RESULT
Antibiotic resistance profile of the clinical isolates
Source In-vitro Antibiotic Resistance
Gram Positive Bacteria
Bacillus subtilis Stool All drug sensitive
Enterococcus faecalis Urine Nx, Ca, Cfm
Micrococcus luteus Throat Swab All drug sensitive
Staphylococcus aureus Sputum Cfx, Cl, Nx, Ca, Cfm, Me
Staphylococcus saprophyticus Urine Cp, Ca, Cfm
Gram Negative Bacteria
Acinetobacter baumannii Urine Cfx, Cl, Ca, Cxm
Citrobacter freundii Urine As, Cfx, Cip, Nx, Of, Sfx, Ca, Cfm
Escherichia coli Urine As, Cfx, Cf, Cl, Nx, Of, Sfx, Ca, Cfm
Klebsiella pneumoniae Urine As, Cfx, Cf, Cl, Mr, Nx, Of, Sfx, Ca, Cfm, Cfs, Ao, Nf
Klebsiella oxytoca Sputum As, Cp, Cfx, Cip, Nx, Of, Ca, Cfm, Cfs, Ao
Moraxella catarrhalis Throat Swab Cfm
Proteus mirabilis Pus Cfx, Do, Lv, Nx, Of, Sfx, Cfm, Ak, Mo
Proteus vulgaris Urine As, Amc, Cfx, Do, Nx, Of, Sfx, Ca, Cfm, Ak, Ao
Pseudomonas aeruginosa Urine As, Amc, Cfx, Do, Nx, Of, Sfx, Ca, Cfm, Cfs, Nf
Shigella flexneri Stool Amp, Ct, T
As: Ampicillin/Sulbactum
(10/10 µg), Amc: Amoxy-
Clavulanic acid (20/10 µg),
Amp: Ampicillin (10 µg), Ct:
Co-trimoxazole
(Sulfamethoxazole/trimethopri
m) (1.25/23.75 µg), Cp:
Cephalexin (30 µg), Cfx:
Cefuroxime (30 µg), Cip:
Ciprofloxacin (5 µg), Cl:
Ceftriaxone (30 µg), Do:
Doxycycline (30 µg), Lv:
Levofloxacin (5 µg), Mr:
Meropenem (10 µg), Nx:
Norfloxacin (10 µg), Of:
Ofloxacin (5 µg), Pt:
Piperacillin/Tazobactum
(100/10 µg), Sfx: Sparfloxacin
(5 µg), Ca: Ceftazidime (30
µg), Cfm: Cefixime (5 µg),
Cfs: Cefoperazone/Sulbactum
(75/30 µg), Ak: Amikacin (30
µg), Ao: Aztreonam (30 µg),
Cxm: Cefotaxime (30 µg), Me:
Methicillin (5 µg), Nf:
Nitrofurantoin (300 µg), T:
Tetracycline (30 µg)

10. RESULT

11. Antifungal activity of the extracts of the stem bark of T. arjuna
*Including diameter of well (6 mm); ATCC: American Type Culture Collection; C.I.: Clinical isolate; hetero-R: hetero resistant; - : No activity.
Fungi (ATCC/C.I.) Source Inhibition zones of
fungal growth (mm*)
Amphotericin B
(0.2 mg/well)
TAW
(MIC)
(mg/mL)
TAM
(MIC)
(mg/mL)
TAW
(1mg/well)
TAM
(1mg/well)
Candida albicans
ATCC 10231
ATCC 15 16 30 0.32 0.16
Candida albicans 1 (C.I.) Sputum 21 20 22 (hetero-R) 0.16 0.16
Candida albicans 2 (C.I.) Sputum 12 10 - 0.64 0.64
Candida tropicalis ATCC 750 ATCC 18 16 24 0.32 0.32
Candida tropicalis 1 (C.I.) Blood 18 19 - 0.32 0.16
Candida tropicalis 2 (C.I.) Blood 20 19 12 0.16 0.32
Candida glabrata (C.I.) Blood 20 18 - 0.16 0.32
Candida krusei (C.I.) Blood 20 21 22 0.16 0.16
RESULT

12. RESULT
Antibacterial activity of the methanol and
water extracts of T. arjuna stem-bark against
ATCC strain of Staphylococcus aureus: (A)
Methanol extract of T. arjuna; (B) Water
extract of T. arjuna; (C) Streptomycin [0.01
mg/well]; (D) Gentamicin [0.01 mg/well];
(E) Penicillin G [0.001 mg/well]; (F) DMSO
Antifungal activity of the water and methanol
extracts of T. arjuna stem-bark against clinical
isolate of Candida krusei: (A) Water extract of
T. arjuna; (B) Methanol extract of T. arjuna;
(C) DMSO; (D) Amphotericin B [0.2 mg/well]

13. RESULT

14. RESULT

15. RESULT
0
1
2
3
4
5
6
Negative
Control
TAW Treated
Cells
TAM Treated
Cells
Positive
Control
Tail
Length
(µm)
0
5000
10000
15000
20000
Negative
Control
TAW
Treated
Cells
TAM Treated
Cells
Positive
Control
Tail
Intensity
(pixels)
0
10
20
30
40
50
60
70
80
90
Negative
Control
TAW Treated
Cells
TAM Treated
Cells
Positive
Control
%
DNA
in
Tail
0
2
4
6
8
10
12
14
16
18
Negative
Control
TAW Treated
Cells
TAM Treated
Cells
Positive
Control
Tail
Moment
Box-whisker plot (graphical representation of the smallest observation, lower
quartile, median, upper quartile, and largest observation) showing comparison
between untreated control, TAW (1 mg/ml) treated, TAM (1 mg/ml) treated, and
100 µL hydrogen peroxide treated fungal cells (positive control) for (A) Tail
length, (B) Tail intensity, (C) %DNA in tail and (D) Tail moment treated fungal
cells.

16. DISCUSSION
• In view of the emerging menace of drug-resistance
pathogens all over the world, it is high time to look
for novel strategies based on traditional plant-based
products to combat microbial infections.
• Previously, Aneja et al. demonstrated the efficacy
of T. arjuna leaf/bark extracts on pathogens causing
ear infection, but not against Candida albicans
(Braz J Otorhinolaryngol, 2012; 78:68-74).
• However, our results have shown pronounced
antifungal activity of TAM/ TAW against several
Candida species including C. albicans, both of
ATCC as well as clinical origin as compared to
Amphotericin B.

17. DISCUSSION
• Again, Singh et al. had tested the inhibitory effect
of triterpenoid constituents of T. arjuna bark, viz.
arjunic acid, arjungenin and arjunetin, against
etiological agents of infectious endocarditis, with no
clear indication about the efficacy against Gram-
negative bacteria (Curr Sci, 2008; 94:27-29).
• Therefore, in the present study it was interesting to
observe a broad-spectrum antibacterial potency of
T. arjuna bark, including the Gram-negative species
in particular.

18. DISCUSSION
• The response of the uropathogens isolated from
patient samples, viz. Enterococcus faecalis,
Staphylococcus saprophyticus, Proteus vulgaris,
Acinetobacter baumannii, Citrobacter freundii,
Escherichia coli, and Pseudomonas aeruginosa,
was particularly encouraging.
• Therefore, it would be worthwhile to explore the
therapeutic value of T. arjuna bark constituents for
application on patients of urinary tract infection,
which is a growing menace particularly among the
female population .

19. DISCUSSION
• Presently, we have shown that only the polar
extracts of T. arjuna bark (TAW and TAM) could
exhibit significant antibacterial activity, irrespective
of the multidrug resistance profile of the individual
pathogenic strains, while TAC was mostly inactive.
Thus, the strong antimicrobial profile of TAM and
TAW might be positively correlated with the
substantial content of polyphenols (~250 – 290 mg
GAE/g) estimated in the polar extracts, and not in
the chloroform extract, viz. TAC.

20. DISCUSSION
• Further, it has been observed that Gram-positive bacteria
would be more susceptible to antimicrobials in
comparison to the Gram-negative bacteria which possess
a thick barrier of lipopolysaccharide layer in the cell
surface. However, the Gram-negative species in our
study were found to respond consistently to TAM/TAW.
• In fact, our revelations on the antimicrobial property of T.
arjuna, a traditionally reputed cardio-protective plant,
assumes added significance in view of the recent concern
about the cardiac safety of antibiotics like
fluoroquinolones, macrolides, trimethoprim–
sulfamethoxazole, etc., on elderly patients susceptible to
cardiac arrhythmias (J Antimicrob Chemother, 2002; 49
:593-596).

21. DISCUSSION
Extent of DNA damage in Candida tropicalis following
treatment with TAW and TAM detected increased levels
of DNA fragmentation in treated fungal cells as
compared with the untreated (negative control) cells (p <
0.05). The study was done in terms of migrated DNA
fragments by using fifteen commonly used parameters,
taking hydrogen peroxide as a standard control.
Interestingly, DNA damage may result in apoptosis or
induction of mutations which are believed to be the mode
of antifungal activity of the said extract.

22. Thank You