In-vitro Antimicrobial Activities of Some Iranian Conifers

Document Type: Research article

Authors

1 Department of Pharmacodynamic and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.

2 Department of Microbiology, Faculty of Medicine, Mashhad, University of Medical Science, Mashhad Iran.

3 Department of Pharmacognosy, Faculty of Pharmacy, Mashhad, University of Medical Science, Mashhad, Iran.

Abstract

Male and female leaves and fruits of eleven different taxons of Iranian conifers (Cupressus sempervirens var. horizontalis, C. sempervirens var. sempervirens, C. sempervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, J. excelsa subsp. excelsa, J. excelsa subsp. polycarpos, J. foetidissima, J. oblonga, J. sabina, Platycladus orientalis and Taxus baccata) were collected from different localities of Iran, dried and extracted with methanol. The extracts were tested for their antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans. The extracts were screened qualitatively using four different methods, the disc diffusion, hole plate, cylinder agar diffusion and agar dilution methods, whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method. The best result was obtained by means of hole plate method in qualitative determination of antimicrobial activities of extracts and the greatest activity was found against S. aureus in all tested methods.

Keywords

Main Subjects


 

 Introduction

    Iranian conifers consist of two families: Cupressaceae and Taxaceae. Cupressaceae consists of one species of Cupressus and one species of Platycladus. The Taxaceae consists of only one species of Taxus.

C. sempervirens, P. orientalisand J. excelsasubspexcelsaare monoecious and others are diecious (1-3).

   An antioxidant activity of methanol extracts of Iranian conifers were investigated previously (4). Cytotoxic study on these plants showed their anti-proliferative activity in cell lines (5-8).

    The progressive resistance of human pathogen microbes against antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP) and vancomycin-resistant Enterococci (VRE), is a growing problem and it is therefore extremely important to find out and develop new antimicrobial compounds. The screening of plant extracts for their antimicrobial activity has shown that higher plants represent a potential source of new anti-infective compounds (9).

    The antimicrobial efficacy of obtained oils from different species of Iranian conifers was showed previously (10-114).

    The purpose of this study is to investigate potential antimicrobial activity of methanol extracts of Iranian conifers, by means of the hole plate, cylinder and disc agar diffusion and agar dilution methods in order to compare the suitability of the screening methods. Whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method.

Experimental

Plant material

    Plant specimens were collected from different parts of the country (Table 1). The plants were identified by Dr. M. Assadi, Research Institute of Forest and Rangelands, Ministry of Jahad Keshavarzi, Iran, Voucher specimens of the taxa have been deposited in the Herbarium of National Botanical Garden of Iran (TARI).

 

Table 1. Plant specimens from different parts of the country.

Plant

Region

Height

Date

Voucher specimen No

C. semipervirens L. var. horizontalis (Mill.) Aiton [syn. C. horizontalis Mill.]

Sorkesh, Aliabad Katool, Golestan province

950 m

2 Oct. 2002

72898

C. semipervirens L. var semipervirens

[syn. C. Pyramidalis Targ.-Tozz]

Ecological Garden of Nowshar, Mazandaran province

23 m

5 Oct. 2002

72890

C. semipervirens L. cv. Cereifeormis

campus of Ferdowsi University, Mashhad, Khorasan Razavi province

920 m

3 March 2003

72893

J. communis L. subsp. hemisphaerica (Presl) Nyman [syn. J. hemisphaerica Presl]

between Damulo and Cephali, Golestan province

2063 m

4 Oct. 2002

72897

J. oblonga M. Beib.

between Makidi and Vainagh, Arasbaran, East Azarbaijan province

1500 m

6 July 2002

72891

J. excelsa M. Beib. subsp excelsa

Kelisa Kharabeh, margin of Aras river, East Azarbayejan province

1400-1600 m

30 Nov. 2002

72895

J. excelsa M.Beib. subsp. polycarpos (K.Koch) Takhtajan [syn. J. polycarpos C. Koch]

Chopoughlou Darahsi, East Azarbaijan province

1593 m

21 Sept. 2002

72900

J. foetidissima Willd.

Makidi and Vainagh, Arasbaran, East Azarbaijan province

1400 m

23 Sept. 2002

72896

J. sabina L.

Sourkesh, Aliabad Katool, Golestan province

2050 m

3 Oct. 2002

No: 72899

P. orientalis (L.) Franco [syn: T. orientalis L.]

Sourkesh, Aliabad Katool, Golestan province

2050 m

2 Oct. 2002

72894

T. baccata L.

Armaniolan, Arasbaran, East Azarbayejan province

1175 m

23 Sept. 2002

72892

 

   The collected materials were stored at -20°C in order to avoid unfavorable changes in the chemical components (15). Extraction of the samples

   Individual fresh leaves of male and female of each plant as well as fruits of them (100 g fresh wt.) were ground by a blinder. Each sample was macerated in pure methanol for 24 h. The samples were then extracted using a percolator. The extracted solutions (27 samples) were concentrated to dryness at 50°C under reduced pressure. The methanol extracts of leaves and fruits of each taxon were evaluated for their antimicrobial activity.

Isolation and Quantification of alkaloids, flavonoids, saponins and tannins

   The fruits and leaves of each plant (500 g) were dried at 50°C and then powdered separately. Each powder was defatted with petroleum ether (bp 40-60°C) using Soxhlet apparatus (6 h). The chemical components of defatted powders were extracted by maceration with methanol (four times). The methanol extracts were concentrated at reduced pressure and the presence of alkaloids (16), flavonoids (17), saponins (18) and tannins (19) were determined (Table 2).

 

 
Table 2. Major components of fruits and leaves of Iranian conifers.
 

Plant name

Plant part

Chemical components (average content)a

 

Alkaloids

Flavonoids

Saponin

Tannins

C. semipervirens var. horizontalis

leaves

2+

4+

3+

C. semipervirens var. horizontalis

fruits

1+

4+

4+

C. semipervirens var. semipervirens

leaves

1+

1+

1+

C. semipervirens var. semipervirens

fruits

1+

2+

C. semipervirens cv. Cereifeormis

leaves

3+

1+

3+

C. semipervirens cv. Cereifeormis

fruits

2+

1+

4+

J. communis subsp. hemisphaerica

leaves (male)

3+

1+

4+

J. communis subsp. hemisphaerica

leaves (female)

3+

1+

4+

J. communis subsp. hemisphaerica

fruits

1+

4+

1+

J. excelsa subsp. excelsa

leaves

1+

2+

 

J. excelsa subsp. excelsa

fruits

2+

1+

1+

J. excelsa subsp. polycarpos

leaves (male)

3+

4+

4+

J. excelsa subsp. polycarpos

leaves (female)

3+

1+

2+

J. excelsa subsp. polycarpos

fruits

2+

1+

2+

J. oblonga

leaves (male)

3+

1+

4+

J. oblonga

leaves (female)

3+

1+

4+

J. oblonga

fruits

3+

1+

2+

J. foetidissima

leaves (male)

4+

3+

J. foetidissima

leaves (female)

4+

2+

J. foetidissima

fruits

4+

2+

J. sabina

leaves (male)

3+

2+

J. sabina

leaves (female)

3+

1+

J. sabina

fruits

P. orientalis

leaves

3+

1+

2+

P. orientalis

fruits

2+

3+

T. baccata

leaves (male)

2+

4+

4+

3+

T. baccata

leaves (female)

3+

2+

3+

T. baccata

fruits

1+

4+

 a: Average content was rated from - to 4+; +: slightly positive; ++: moderately positive; +++: strongly positive; ++++: very strongly positive; -: not detected.

 

Test organisms

    The following microbial strains for testing purposes were purchased from the Persian Type Culture Collection (PTCC): Escherichia coli PTCC 1330, Staphylococcus aureus PTCC 1337, Pseudomonas aeruginosa PTCC 1074, and Candida albicans PTCC 5027.

    The negative and positive controls were respectively methanol and antibiotics containing discs (gentamycin 10 μg/disc and clotrimazole 8 μg/disc).

Hole diffusion method

   This assay was performed using a suspension with 0.5 McFarland standard turbidity. Holes of 6 mm diameter were then made on the Mueller Hinton agar (Merck) plate (8 mm thick) inoculated by flooding and filled with 50 μL of methanol extract. The plates containing the bacteria and C. albicans were respectively incubated at 37°C and 25°C for 24 and 48 h. The antimicrobial activity was evaluated by measuring the inhibition zone (IZ) around each hole. They were recorded as (-) for non-active samples and (+) for samplespresenting IZ greater than 6 mm (the diameter of the hole) (20).

Cylinder plate diffusion test

  This method is the same as hole diffusion test except that the filled cylinder containing 200 μL of different concentration of each extract used as the filled holes. The negative and positive controls were methanol and solutions of two antibiotics (gentamycin 0.2 μg/mL and clotrimazole 0.16 μg/mL), respectively (21).

Disc diffusion method

   This assay was performed using the filter paper disc diffusion method on Mueller Hinton and sabouraud dextrose agar respectively for bacteria and C. albicans. The plates were incubated under sterile conditions at 37°C for 24 h. Inoculums were prepared using a suspension with 0.5 McFarland standard turbidity and the culture was spread over the plates by means of a sterile cotton swab.

   Plant extracts (0.25-2 mg/disc) were prepared and placed on plates earlier inoculated with microbial suspension. This was done to evaluate the sensitivity of the extracts at which microbial growth was inhibited effectively (22).

Agar well dilution test

   Briefly, the methanol extracts (100, 50, 25 and 12.5 μg/mL) were diluted in molten Mueller Hinton agar (MHA, Merck) on 24 well plates. All bacterial strains were grown in Mueller Hinton broth (MHB, Merck) for 4 h at 37°C. Bacterial suspensions with 0.5 McFarland standard turbidity (≈108 cfu/mL), were prepared by dilution with Mueller Hinton broth. The diluted inoculums were added to a Steer’s replicator calibrated and incubated for 24 h at 37°C and 48 h at 25°C respectively for bacteria and C. albicans (23).

Results and Discussion

   The antimicrobial activities of 27 methanol extracts of different parts of Iranian conifers were determined using four different methods. Screening was carried out at four different concentrations against S. aureus, E. coli, P. aeruginosa and C. albicans strains to examine the sensitivity against the mentioned micro-organism. Table 3 shows the most sensitive microbial strains in different methods.

 

 

Table 3. Most sensitive microbial strains in different methods.

 

Determination of micro-organism sensitivity in diffusion methods

   Table 4 shows the antimicrobial activity of Iranian connifer extracts (25, 50, 100 and 200 mg/mL) in three different methods. Methanol extracts exhibited only weak or no activity in cylinder agar diffusion method perhaps because of the low extracts diffusion in agar or may be the precipitation of insoluble material that inhibits further diffusion of active constituent. In other methods, the extracts exhibited significant growth inhibition on S. aureus. In hole plate method, a weak antibacterial activity against E. coli was assessed whereas it showed complete resistance in disc diffusion method as C. albicans did. P. aeruginosa’s growth was inhibited 62% and 11.1% in hole plate and disc diffusion methods respectively. The extracts were 11.1% effective on C. albicans in hole plate assay.

 

 

Table 4. Antimicrobial activity of Iranian connifer extracts for concentration of 25, 50, 100 and 200 g L-1 in three different methods. 


a: Con = Concentration; b = DDM: Disc diffusion method; c : HDP = Hole plate difffusion; d : CAD = Cylinder agar diffusion; e: absence of inhibition zones; f: Inhibition zone ≥ 6 showed the antimicrobial activity; g: positive control; Data are shown as mean ± SD of three independent experiments.

C1 = C. semipervirens var. horizontalis leaf, C2 = C. semipervirens var. horizontalis fruit, C3 = C. sempervirens leaf, C4 = C. sempervirens fruit, C5 = C. sempervirens. cv. Ceriformis leaf, C6 = C. sempervirens. cv. Ceriformis fruit, C7 = J. communis subsp. hemisphaerica female leaf, C8 = J. communis subsp. hemisphaerica male leaf, C9 = J. communis subsp. hemisphaerica fruit, C10 = J. excelsa subsp. excels leaf, C11 = J. excelsa subsp. excelsa F, C12 = J. excelsa subsp. polycarpos female leaf, C13 = J. excelsa subsp. polycarpos male leaf, C14 = J. excelsa subsp. polycarpos fruit, C15 = J. foetidissima female leaf, C16 =J. foetidissima male leaf, C17 = J. foetidissima fruit, C18 = J. oblonga fL, C19 = J. oblonga male leaf, C20 = J. oblonga fruit, C21 = J. sabina female leaf, C22 =J. sabina male leaf, C23 = J. sabina fruit, C24 = P. orientalis leaf, C25 = P. orientalis fruit, C26 = T. baccata female leaf, C27 = T. baccata male leaf.

 

   The micro-organism sensitivity in low (25 μg/mL) and high (200 μg/mL) concentrations of extracts were differing in hole plate method as shown in Table 5.

 

Table 5. Comparison of the micro-organism sensitivity in low (25 μg/mL) and high (200 μg/mL) concentrations in hole plate method.

Extract concentration

Sensitivity

(25 μg/mL)

S. aureus < P. aeruginosa < C. albicans < E. coli

(200 μg/mL)

S. aureus < C. albicans < P. aeruginosa < E. coli

 

MIC of each extracts as determined by agar diffusion method

   The MIC exhibited by the Iranian conifer extracts against tested bacterial and fungal strains are given in Table 6. Obtained results revealed that the extracts are generally more active on S. aureus and P. aeruginosa whereas C. albicans and E. coli had higher MIC values.

 

 

Table 6. Minimal inhibitory concentration (mg/mL) of the methanol extracts of different parts of Iranian conifer species with agar dilution test.

Plant extract

Used part

C. albicans

E. coli

P. aeruginosa

S. aurus

C. sempervirens var. horizontalis

leaf

100

50

12.5

0.39

fruit

50

50

25

0.78

C. sempervirens var. sempervirens

leaf

50

100

25

1.56

fruit

25

50

25

1.56

C. sempervirens cv. Cereifeormis

leaf

12.5

12.5

12.5

0.78

fruit

50

50

12.5

0.78

J. communis subsp. hemisphaerica

female leaf

50

50

25

0.39

male leaf

50

50

25

0.78

fruit

50

50

25

0.39

J. excelsa subsp. excelsa

leaf

12.5

25

12.5

0.39

fruit

50

100

25

0.78

J. excelsa subsp. polycarpos

female leaf

25

50

12.5

0.39

male leaf

12.5

25

12.5

0.39

fruit

50

50

25

0.78

J. foetidissima

female leaf

25

50

12.5

0.78

male leaf

25

50

12.5

0.78

fruit

25

50

25

0.78

J. oblonga

female leaf

50

50

25

0.78

male leaf

25

50

12.5

0.39

fruit

50

100

25

3.12

J. sabina

female leaf

50

50

12.5

0.78

male leaf

50

50

12.5

0.78

fruit

50

50

12.5

0.78

P. orientalis

leaf

25

500

25

0.39

fruit

50

50

25

1.56

T. baccata

female leaf

25

6.25

12.5

3.12

male leaf

50

12.5

12.5

3.12

            MIC values in mg/mL of extracts and antibiotics. Gentamicin and clotrimazole used as positive controls.

 

 

Antimicrobial effects of the investigated Cupressus species

   All of the three tested cupressus taxa had antimicrobial properties against S. aurus. They have weak or no activity on other strains and this result, except for S. aurus, is in agreement with the literature. Alkofahi et al. and Guerinet al. (24, 25) reported that extracts of C. sempervirens: showed no activity against tested strains. The contradictory about S. aurus might be due to the extract concentrations, microbial strains or solvent used in our study. Methanol extracts had the most marked antimicrobial effects of all the tested species in different studies (26). C. sempervirens and C. sempervirens. cv. Cereiformis have been found to possess antimicrobial activities against P. aeroginosa in our study. It is believed that this effect shown by these taxa is due to the limitations discussed above. Essential oil of C. sempervirens was shown to be a potent inhibitor of S. aurus and P. aeroginosa, (27) therefore, the presence of essential oil constituent in extracts can be assumed. Since it is known that the leaves and fruits of cupressus species contain Camphen, Quercetin, Catechin, Linalool, Borneol and Sabinen, a part of the antimicrobial activities we investigated might be due to these constituents (28-30).

Antimicrobial effects of the investigated Juniperus species

   As Dornberger et al. (31) had found antimicrobial properties in J. communis, and Muhammad et al. and Topcu et al. (32, 33) found evidence of antibacterial activity from the leaves and fruit of J. excelsa, it was not a surprise tofind antimicrobial extracts among the Juniperus species investigated.

   Methanol leaves and fruits extracts of all Juniperus species were effective against S. aureus by hole plat method. All of the Juniperus species leaves extracts were shown to be potent inhibitor of P. aeroginosa.

   Extracts of J. communis subsp. hemisphaerica proved to be effective against C. albicans in high concentration with hole plate and among those, female leave extract revealed the greatest activity. The antibacterial effects against P. aeroginosa were only shown in the female leave extract by disc diffusion method.

   Methanol fruits extract of J. excelsa were effective against C. albicans which have been investigated by Topcu et al. (33) previously.

   The antibacterial activity against P. aeroginosa, which have been investigated earlier by H24107, was shown in the leave extract.

    Except the fruits extract, the other parts of J. excelsa subsp. polycarpos were found to be effective against P. aeroginosa.

    Methanol extracts of fruits and leaves of J. foetidissima and fruit and female leave of J. oblanga were effective against all of the tested microbes by hole plat method. A methanol extract of J. foetidissima and J. oblanga was added to the tested Juniperus extracts to inhibit the growth of E. coli.

Antimicrobial effects of the investigated Platycladus orientalis

    Like other species, methanol extracts of leaves and fruits of P. orientalis were effective against S. aureus by hole plat method. The antimicrobial activities of methanol extracts of P. orientalis did not differ to a marked extent in hole plate method, and were all inhibitors of all the micro-organisms except for E. coli. Our antimicrobial results justify the Dornberger et al. findings (31). They were only effective against S. aureus in disc diffusion method.

Antimicrobial effects of the investigated Taxus baccata

    Like what stated previously, methanol extracts of leaves and fruits of T. baccata were effective against S. aureus by hole plat method. Our finding about methanol extracts of T. baccata revealed that all of the microbial strains were sensitive except for C. albicans. By disc diffusion method, S. aureus was the only micro-organism that showed growth inhibition like that obtained with P. orientalis.

Minimum inhibitory concentration values of the extracts

    Methanol extracts of the all taxa gave lowest MIC-values against S. aureus (< 3.12 mg/mL) compared to the other strains which had rather higher MIC-values. The reasons for the high MIC values could be that the extracts are mixtures of a large number of compounds and they might suppress the biological activities of each other, or that the active compound(s) is present in very low concentrations. Furthermore, plant extracts generally contain secondary metabolites like saponins, terpenoids and phenolics in a physiologically inactive glycoside form, and this may explain why some of the extracts did not produce very marked inhibition (34). Other reasons could be the slow diffusion of large molecules into the agar and masking detection of the full antimicrobial potential of the extract. This could be overcome by using the turbidimetric method, but there are several problems associated with this method while studying the bioactivities of crude extracts containing large molecules like tannins (35). One major problem is the formation of precipitation in tannin containing crude extracts, which makes it impossible to use turbidity as a measure of bacterial growth. This can be overcome by using INT (p-iodonitrotetrazolium violet) which is reduced to the red colored product formazan indicating bacterial growth (36).

   Of the 27 extracts of four conifer species we investigated, the most antimicrobial potent one was a leaf extract in methanol of C. sempervirens. cv. Ceriformis which had the lowest MIC in comparison with other extract on four strains. Most of the tested plants showed the antimicrobial activity to some extent. It is possible that these essential oils from coniferous trees can be used as antibacterial and/or antifungal agents in food or other ingredients. The mechanism of antibacterial and antifungal effects of these extracted from coniferous trees needs to be further examined for potential uses. These results indicate that the essential oils derived from coniferous trees, which have mild antimicrobial properties, can inhibit the growth of Gram-positive and Gram-negative bacteria and fungi.

Acknowledgements

   This work was supported by grants from Research Affairs of Mashhad University of Medical Sciences, the specialized Research fund for the Pharmacy Doctoral Program.

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