Clinical isolates of urinary tract infection and candidiasis inhibited by T. arjuna bark extract
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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.
![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]](https://image.slidesharecdn.com/deyetal-210724053843/85/Clinical-isolates-of-urinary-tract-infection-and-candidiasis-inhibited-by-T-arjuna-bark-extract-12-320.jpg)
Clinical isolates of urinary tract infection and candidiasis inhibited by T. arjuna bark extract
- 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