Antifungal and antibacterial activities of Taxus wallichiana Zucc

Abstract

Current study was undertaken to evaluate the in vitro antifungal and antibacterial potential of methanol extract and subsequent fractions obtained after partitioning in organic solvents with variable polarity of the aerial parts of the tree Taxus wallichiana Zucc. Traditionally, this plant is often used in folk medicines in Pakistan for treating microbial infections. In order to rationalize the traditional use, methanol extracts of leaf, bark, and heartwood of Taxus wallichiana Zucc. were tested against six bacteria and six fungal strains using the Hole diffusion and macro-dilution methods. All extracts and fractions displayed significant antimicrobial effect. Only three fungal strains, Trichophyton longifusus, Microspoum canis, and Fusarium solani were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. Comparison results were carried out using imipinem, miconazole and amphotericin B as standard antibiotics.

Keywords::

• Taxus wallichiana zucc
• antifungal
• antibacterial

Introduction

Globally, infectious disease is the major cause of death, accounting for approximately one-half of all deaths, in tropical countries [1]. New therapeutic agents and strategies are demanding issues to cope with infectious diseases. Low-income people especially from small isolated villages and native communities use folk medicine for the treatment of common infections. These plants are ingested as decoctions, teas and juice preparations to treat respiratory infections or as a poultice and applied directly on the infected wounds or burns 2-3.

Taxus wallichiana Zucc. is a member of the family Taxaceae which is commonly known as the Himalayan yew [4]. The plant is widely distributed in Pakistan and India, and used in the traditional systems of medicine [5]. Taxol and related bioactive taxoids have been reported from the various species of the genus, including Taxus wallichiana Zucc. 6-8. Other pharmacological activities are also reported from the plants of genus Taxus which includes, imunomodulation [9], antiallergic activity [10], antinociceptive and antinflammatory activity [11], antiosteoporotic activity [12], antiplatelet activity and vasorelaxing effect [13], DPPH radical scavenging and nitric oxide (NO) inhibitory activities [14].

Antifungal activities of various taxoids isolated from Taxus cuspidata var. nana has been reported against plant pathogenic fungi [15]. In another study, Taxus baccata heartwood extract showed significant activity against selected gram-negative bacteria and selected pathogenic fungi [16]. Bilobetin, a biflavone from needles of Taxus baccata, has been reported to possess the significant antifungal activity [17]. In Pakistan, this plant is used traditionally for treatment of pyrexia, acute painful conditions, indigestion, epilepsy and for the treatment of wounds and skin infections [3,5,18]. However, thorough literature survey has revealed that no significant work has been done on antibacterial and antifungal activities of the Taxus wallichiana Zucc. Keeping this knowledge in view, the present study was undertaken to investigate the antibacterial and antifungal potential of Taxus wallichiana Zucc. We report here the results of antimicrobial testing of Taxus wallichiana Zucc. extract against Escherchia coli, Bacillus subtilis, Shigella flexeneri, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. Fungal strains includes Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata

Materials and methods

Plant material

Plant material was collected from the Hazara division of the North-western Frontier Province, Pakistan, in March 2005 and identified by a taxonomist, Dr. Hasan Sher, Department of Botany, Jehanzeb Postgraduate College Saidu Sharif, Swat. A voucher specimen was deposited in the herbarium of the same institution. The aerial parts of the plant were air-dried under shade for six consecutive weeks at room temperature. The dried plant material was later on chopped, finely ground and stored in a polyethylene bag under refrigeration for further experimentation.

Extract preparation

The dried and powdered leaves, bark and wood (2.5 Kg, 4.0 Kg and 1.0 kg) respectively) were macerated in methanol with occasional manual shaking at room temperature for a period of 72 h. After filtration, the process was repeated 3 times using additional methanol each time. The combined filtrates were evaporation under reduced pressure at 40° C. The concentrated methanol extracts of leaves (357 g, 14.28% w/w), bark (514 g, 12.85% w/w), and wood (94 g, 9.4% w/w) were obtained. Crude methanolic extract of bark was redissolved in distilled water and successively extracted with hexane (11%), chloroform (31.9% w/w), ethyl acetate (38.8% w/w), and finally water (18.2% w/w) to give the respective fraction. Each organic extract was then evaporated to dryness. Stocks extract solutions were prepared at 200 mg/mL in distilled water. The pH was adjusted between pH 5–7. Extracts were sterilized over a membrane filter unit of 0.2 μm of pore size (Minisart Sartorius) and preserved at +4°C until used.

Fungal and bacterial strains

Tests were performed on six fungi and six bacteria reference strains. Bacterial strains were E. coli ATCC 25922, B. subtilis ATCC 6633, S. flexeneri (clinical isolate), S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and S. typhi ATCC 19430. Fungal strains include T. longifusus (clinical isolate), C. albicans ATCC 2091, A. flavus ATCC 32611, M. canis ATCC 11622, F. solani 11712 and C. glaberata ATCC 90030. They were maintained on an agar slant at 4°C. The strains were activated at 37°C for 24 h on nutrient agar (NA) or Sabouraud glucose agar (SGA) respectively for bacteria and fungi, prior to any screening.

Hole-diffusion method

The antimicrobial tests were carried out by the hole-diffusion method using a cell suspension of about 1.5 × 106 CFU/mL obtained following Macfarland turbidity standard No. 0.5 [19]. The concentration of the suspension was standardized by adjusting the optical density to 0.1 at 600 nm (Shimadzu, UV-VIS Spectrophotometer) [20]. Holes of 6 mm diameter were then made on the MHA plate (8 mm thick) and filled with 150 μL of methanolic extract, fractions or standard drug(s). The inoculated plates were incubated at 37°C for 24 h. Antimicrobial activity was evaluated by measuring the diameter of the zone of growth inhibition around the hole. The assay was repeated three times and the mean diameter was recorded. Imipenem, miconazole and amphotericin B were used as standard antibiotics for comparison with extracts and fractions.

MIC determination by macrodilution method

Extracts (10 mg/mL) were dissolved in DMSO and serially diluted with sterile water in microplates in a laminar flow cabinet. The same volume of an actively growing culture of the test bacteria was added to the different wells and cultures were grown overnight in 100% relative humidity at 37°C. Next morning, tetrazolium violet was added to all the wells and the growth was indicated by a violet color of the culture. The lowest concentration of the test solution that led to inhibition of growth was taken as the MIC. The negative control acetone had no influence on the growth even at the highest concentration used. Imipenem, amphotericin B and miconazole were used as controls for comparison.

Results

Using the hole diffusion and macrodilution technique (Table I), leaf extracts of Taxus wallichiana Zucc showed antimicrobial properties with MIC values ranging from 0.23 to 200 mg/mL for bacterial strains and 0.11 to 200 mg/mL for fungi. Taxus wallichian extracts show the lowest MIC values (Table II) and thus they may warrant the presence of interesting antimicrobial lead compounds.

Table I.  In vitro antibacterial activity of crude extracts and fractions of Taxus wallichiana Zucc. Hole-diffusion method.

Name of Bacteria	Zones of inhibition of bacterial growth (in mm) by various samples
	Std. drug	IB	IL	IW	IF-1	IF-2	IF-3	IF-4
Escherchia coli	24	10	9	10	–	–	10	–
Bacillus subtilis	23	–	–	–	–	–	–	–
Shigella flexeneri	28	–	–	–	–	–	–	–
Staphylococcus aureus	27	12	–	11	–	9	–	10
Pseudomonas aeruginosa	20	14	14	17	–	–	14	15
Salmonella typhi	26	13	12	–	–	–	–	12

Std. drug: imipenem, IB: Methanol bark extract, IL: Methanol leaf extract, IW: Methanol bark extract, IF-1: n-Hexane fraction of IB, IF-2: Chloroform fraction of IB, IF-3: Ethyl acetate fraction of IB and IF-4: Water fraction of IB.

Table II.  In vitro antifungal activity of crude extracts and fractions of Taxus wallichiana Zucc. Hole-diffusion method.

Bacteria	% Inhibition of Fungal Growth By Various Samples
	Std. drug	IB	IL	IW	IF-1	IF-2	IF-3	IF-4
Trichophyton longifusus	70^1	30	60	50	40	–	50	–
Candida albicans	110^1	–	–	–	–	–	–	–
Aspergillus flavus	20^2	–	–	–	–	–	–	–
Microsporum canis	98.4^1	20	40	50	80	–	–	–
Fusarium solani	73.26^1	80	–	60	90	90	–	–
Candida glaberata	110.8^1	–	–	–	–	–	–	–

1 Standard Drug = Miconazole,

2 Standard Drug = Amphotericin B; IB: Methanol bark extract, IL: Methanol leaf extract, IW: Methanol bark extract, IF-1: n-Hexane fraction of IB, IF-2: Chloroform fraction of IB, IF-3: Ethyl acetate fraction of IB and IF-4: Water fraction of IB.

In vitro antibacterial activity of extracts

Methanol extract of Taxus wallichiana Zucc. bark, leaves and wood (IB, IL and IW), ethyl acetate fraction of the former bark extract (IF-3) and the aqueous fraction of methanol bark extract (IF-4) indicated lowest MIC and inhibited the bacterial strains, S. aureus, P. aeruginosa and S. typhi (Table I, III). Among others, the IB remained the most active extract with MIC = 0.61, 0.49, 0.31 and 0.42 mg/mL for E. coli, S. aureus, P. aeruginosa and S. typhi, respectively). E. coli and P. aeruginosa were the most susceptible bacteria with MIC ranging from 0.23 mg/mL to 0.67 mg/mL.

Table III.  Antibacterial Activity of Crude Extracts and Fractions by macrodilution method.

Bacterium	MIC (mg/mL)
	Std. drug	IB	IL	IW	IF-1	IF-2	IF-3	IF-4
Escherchia coli	0.0002	0.83	0.67	0.62	>200	50	0.64	>200
Bacillus subtilis	0.0005	>200	>200	>200	>200	>200	>200	>200
Shigella flexeneri	0.0003	150	150	>200	50	>200	>200	>200
Staphylococcus aureus	0.0009	0.49	150	0.54	>200	0.59	50	0.61
Pseudomonas aeruginosa	0.0021	0.31	0.33	0.23	50	>200	0.39	0.26
Salmonella typhi	0.0014	0.42	0.46	>200	>200	>200	>200	0.38

Std. drug: imipenem, IB: Methanolic bark extract, IL: Methanolic leaves extract, IW: Methanolic bark extract, IF-1: Hexane fraction of IB, IF-2: Chloroform fraction of IB, IF-3: Ethyl acetate fraction of IB and IF-4: Water fraction of IB.

In vitro antifungal activity of extracts

Taxus wallichiana Zucc.extracts exhibited from significant to remarkable inhibitory activities against T. longifusus, M. canis, and F. solani, except the aqueous fraction of methanol bark extract (IF-4) which did not exhibit any noticeable action against these strains. Crude extracts and the fractions showed a zone of inhibition (in mm) against these strains (Table II). As regards the selective inhibitory role of extracts and the polar fractions of the plant under investigation, the species F. solani was strongly inhibited with MIC ranging from 0.08 mg/mL to 200 mg/mL, followed by the M. canis with MIC ranging from 0.17 mg/mL to 200 mg/mL and then T. longifusus (MIC ranging from 0.19 mg/mL to 200 mg/mL. (Table IV) Interestingly, all the extracts show negligible activity against C. albicans, A. flavus and C. glaberata.

Table IV.  Antifungal Activity of Crude Extracts and Fractions by macrodilution method.

Fungus	MIC (mg/mL)
	Std. drug	IB	IL	IW	IF-1	IF-2	IF-3	IF-4
Trichophyton longifusus	0.0014^1	>200	0.19	0.22	0.28	3.54	0.23	>200
Candida albicans	0.0001^1	>200	100	>200	100	150	>200	>200
Aspergillus flavus	0.027^2	>200	>200	>200	>200	>200	>200	>200
Microspoum canis	0.0006^1	0.78	0.32	0.21	0.17	50	>200	>200
Fusarium solani	0.0011^1	0.11	50	0.24	0.09	0.08	150	>200
Candida glaberata	0.0003^1	100	100	100	>200	>200	>200	>200

1 Standard Drug = Miconazole,

2 Standard Drug = Amphotericin B IB: Methanolic bark extract, IL: Methanolic leaves extract, IW: Methanolic bark extract, IF-1: Hexane fraction of IB, IF-2: Chloroform fraction of IB, IF-3: Ethyl acetate fraction of IB and IF-4: Water fraction of IB.

Discussion

The methanol extract of the aerial parts and the polar fractions thereof of Taxus wallichiana Zucc were found to possess inhibition activity against seven representative pathogens studied, which strongly supports the traditional use for gastroenteritis and skin related diseases. The MICs of the extracts observed against the sensitive strains ranged from 0.23 to 200 mg/mL (for bacterial strains) and 0.08 to 200 mg/mL (for fungal strains). Furthermore, a gradual increase in the antimicrobial activity was observed against the tested pathogens when progressing from the crude extract to polar fractions. This gradual increase in activity proved successful with both procedures employed i.e. hole diffusion and the macro-dilution techniques. This was mainly observed with the MICs obtained with extracts and fractions on T. longifusus, M. canis, and F. solani. However, in case of bacterial strains, IW and IF-4 showed potent activity against the P. aeruginosa having MICs 0.23 and 0.26 mg/mL. IL, IW, IB and IF-4 exhibited significant activity against the E. coli having MICs 0.62, 0.67, 0.83 and 0.64 mg/mL respectively. Similarly, IW, IB, IF-2 and IF-4 displayed significant activity against the S. aureus having MICs of 0.54, 0.49, 0.59 and 0.61 mg/mL. This antimicrobial activity may be attributed to the presence of alkaloids, phenols, polyphenols, saponins, tannins, anthraquinones, steroids and especially the diterpenes, found in the crude extract and the fractions thereof. These phytochemical groups/families of natural products are known to display antimicrobial activities 21-27. Further purification and characterization of the active principles from fractions IF-1 and IF-2 (for antibacterial studies), IF-3 and IF-4 (for antibacterial studies) will provide a better understanding of the antimicrobial mechanism and may serve as a tool for isolation of potential lead compounds for microbial infectious diseases.

Acknowledgements

We thank Dr. Hasan Sher, Department of Botany, Jehanzeb Postgraduate College Saidu Sharif, Swat, Pakistan for providing technical assistance in collection and authentication of the plant. This project was supported jointly by the University of Peshawar and the H.E.J.Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi-75270, Pakistan.