Antimicrobial Studies on Extracts of Three Species of Phlomis.

Abstract

The antimicrobial activity of Phlomis bruguieri. Desf., P. herba-venti. L., and P. olivieri. Benth. (Labiatae), native plants of Iran, were studied using the disk diffusion method and determination of minimum inhibitory concentration (MIC) values against Staphylococcus aureus. PTCC 1112, Streptococcus sanguis. PTCC 1449, Escherichia coli. PTCC 1330, Pseudomonas aeruginosa. PTCC 1074, Klebsiella pneumoniae. PTCC 1053, Aspergillus niger. PTCC 5011, and Candida albicans. PTCC 5027. The methanol extracts of the aerial parts of Phlomis bruguieri., P. herba-venti., and P. olivieri. exhibited concentration-dependent antibacterial activity against all tested bacteria. The methanol extracts were more active against Gram-positive microorganisms (Streptococcus sanguis. and Staphylococcus aureus.). The extracts did not show antifungal activity.

Keywords:

• Antimicrobial activity
• Labiatae
• Phlomis bruguieri
• P. herba-venti
• P. olivieri

Introduction

The genus Phlomis. (Labiatae) comprises 17 species that are widely distributed in the Azerbaijan, Fars, Gilan, Hamedan, Isfahan, Kurdestan, and Mazandaran provinces of Iran (Rechinger, 1982; Mozaffarian, 1996). Several Phlomis. species are used in herbal medicine; for example, for diseases of the respiratory tract or externally for treatment of wounds (Morteza-Semnani & Saeedi, 2005). Plants belonging to the genus Phlomis. have been shown to contain iridoid glucosides, flavonoid glycosides, phenylethanoid glycosides, diterpene glycosyl esters, and nortriterpenes (Couladis et al., 2000; Kamel et al., 2000).

Phlomis bruguieri. Desf., P. herba-venti. L., and P. olivieri. Benth. are aromatic plants native to Iran (Mozaffarian, 1996). We have already reported the essential oil composition of Phlomis bruguieri. and P. herba-venti. (Morteza-Semnani et al., 2004; Morteza-Semnani & Saeedi, 2005). In a recent study, germacrene D (23.6%), 4-hydroxy-4-methyl-2-pentanone (15.0%), α.-pinene (6.8%), and β.-caryophyllene (6.7%) were the major constituents of the oil of Phlomis bruguieri. collected from the suburb of Larijan, Mazandaran province, northern Iran (Morteza-Semnani & Saeedi, 2005). In 2004, the oil compositions of the leaves and flowers of P. herba-venti. L. collected from the suburb of Sari, Mazandaran province, northern Iran, was reported; the major constituents of the leaf oil were germacrene D (33.9%), hexadecanoic acid (12.9%), and α.-pinene (9.4%), and the main components of the flower oil were hexadecanoic acid (33.1%), 6,10,14-trimethylpentadecan-2-one (16.2%), 3-methyltetradecane (6.7%), and germacrene D (6.7%) (Morteza-Semnani et al., 2004).

The total extract of P. olivieri. showed an antinociceptive effect using the visceral writhing test model in mice (Sarkhail et al., 2003).

There is no report on the antimicrobial activity of these plants. The medicinal properties attributed to the genus Phlomis. prompted us to investigate the antimicrobial activity of the methanol extracts of Phlomis bruguieri., P. herba-venti., and P. olivieri. for the first time.

Materials and Methods

Materials

The flowering aerial parts of Phlomis bruguieri. were collected in May 2004 from the suburb of Larijan, Mazandaran province, northern Iran, and the flowering aerial parts of P. herba-venti. and P. olivieri. were collected in May 2004 from the suburb of Sari, Mazandaran province, northern Iran, and identified by Mohammad Akbarzadeh (Department of Botany, Research Center of Natural Resources of Mazandaran). Voucher specimens (herbarium no. 115, 118, and 119) were deposited in the herbarium of the Department of Botany, Research Center of Natural Resources of Mazandaran.

Extraction

Dried plant materials (100 g) were ground to fine powder and extracted twice with methanol. The extacts were evaporated to dryness at 40°C and stored in a refrigerator.

Bioassay

In vitro. antimicrobial studies were carried out by the disk diffusion method, and minimum inhibitory concentration (MIC) values were determined against test microorganisms (Baron & Finegold 1990; Awadh Ali et al., 2001). In the disk diffusion method, extracts were dissolved in methanol and applied to a 6-mm-diameter paper disk. The extracts were tested at 10, 50, 100, 250, 500, 750, and 1000 µg/disk. Inhibition zone diameters were measured after 24 h. Gentamicin (50 µg/disk), amikacin (3 µg/disk), and amphotericin B (100 µg/disk) (obtained from Sigma, Germany) were used as positive controls.

MICs were determined by the dilution method at concentrations of 10 µg/ml to 25 mg/ml of culture medium (Morteza-Semnani et al., 2005). Gentamicin (2 mg/ml) and amphotericin B (100 µg/ml) were used as positive controls.

Staphylococcus aureus. PTCC 1112, Streptococcus sanguis. PTCC 1449, Escherichia coli. PTCC 1330, Pseudomonas aeruginosa. PTCC 1074, Klebsiella pneumoniae. PTCC 1053, Aspergillus niger. PTCC 5011, and Candida albicans. PTCC 5027 were used for testing the antimicrobial activity.

Results and Discussion

The yields of methanol extracts of Phlomis bruguieri., P. herba-venti., and P. olivieri. were 10.6%, 11.3%, and 9.2%, respectively.

Tables 1 and 2 give a summary of the Phlomis. species investigated and the results of the antimicrobial screening. The methanol extracts of the dried flowering aerial parts of Phlomis bruguieri., P. herba-venti., and P. olivieri., exhibited concentration-dependent antibacterial activity against all tested bacteria. The methanol extracts were more active against Gram-positive microorganisms (Streptococcus sanguis. and Staphylococcus aureus.). The methanol extracts were found to be most effective against Streptococcus sanguis.. The extracts did not show antifungal activity against fungi.

Table 1. Antimicrobial activity of the methanol extracts of Phlomis bruguieri., P. herba-venti., and P. olivieri.^a..

		Diameter of zone of inhibition (mm)
		Bacteria					Fungi
Sample	Concentration (µg/disk)	Staphylococcus aureus. (G+)	Streptococcus sanguis. (G+)	Escherichia coli. (G−)	Pseudomonas aeruginosa. (G−)	Klebsiella pneumoniae. (G−)	Candida albicans.	Aspergillus niger.
P. bruguieri.	10	10.1	13.8	–	–	–	–	–
	50	11.2	14.2	–	–	–	–	–
	100	12.1	15.0	–	–	9.8	–	–
	250	13.4	16.0	–	–	12.2	–	–
	500	14.8	16.8	9.2	9.9	13.1	–	–
	750	15.6	17.7	9.9	10.9	14.8	–	–
	1000	16.7	18.6	11.1	12.3	15.8	–	–
P. herba-venti.	10	–	–	–	–	–	–	–
	50	–	–	–	–	8.7	–	–
	100	–	8.3	–	–	10.5	–	–
	250	8.7	9.3	8.4	–	11.8	–	–
	500	9.9	10.6	9.2	8.5	13.2	–	–
	750	11.1	11.8	10.2	9.3	13.9	–	–
	1000	12.2	13.3	11.2	10.9	14.6	–	–
P. olivieri.	10	–	–	–	–	8.9	–	–
	50	–	–	–	–	9.8	–	–
	100	–	–	–	–	10.9	–	–
	250	8.9	–	9.0	–	12.3	–	–
	500	10.3	8.9	10.4	8.9	13.3	–	–
	750	11.5	10.6	11.5	9.8	14.3	–	–
	1000	13.1	12.0	13.6	11.5	15.9	–	–
Gentamicin	50	37.3	24.0	31.6	31.0	28.0	–	–
Amicacin	3	24.9	19.9	23.8	15.8	18.0	–	–
Amphotericin B	100	–	–	–	–	–	22.3	22.7

^a.Zone of inhibition, including the diameter of the filter paper disk (6 mm); mean value of eight independent experiments; gentamicin, amikacin, and amphotericin B were used as positive controls; −, no inhibition.

Table 2. Minimum inhibitory concentration (MIC) of Phlomis bruguieri., P. herba-venti., and P. olivieri.^a..

	MIC (mg/ml)
	Bacteria					Fungi
Sample	Staphylococcus aureus. (G+)	Streptococcus sanguis. (G+)	Escherichia coli. (G−)	Pseudomonas aeruginosa. (G−)	Klebsiella pneumoniae. (G−)	Candida albicans.	Aspergillus niger.
P. bruguieri.	10	1	25	25	25	–	–
P. herba-venti.	10	25	25	40	25	–	–
P. olivieri.	25	10	10	25	10	–	–

^a.All determinations were done in triplicate; gentamicin (2 mg/ml) and amphotericin B (100 µg/ml) were used as positive control.

The essential oils and ethanol exract of P. fruticosa. L. were evaluated for antibacterial and antifungal activities by Ristic et al. (2000). The essential oils showed antibacterial activity against Staphylococcus aureus., Escherichia coli., Bacillus subtilis., Klebsiella pneumoniae., and Micrococcus luteus.. The essential oils extracted from the plants collected from two different localities showed similar antibacterial activities. The antifungal activity of the essential oils was positive against Aspergillus niger., A. ochraceus., Cladosporium cladosporioides., Fusarium tricinctum., and Phomopsis helianthi.. The ethanol extract showed antibacterial activity against Staphylococcus aureus. and Bacillus subtilis. and antifungal activity against Aspergillus niger., A. ochraceus., Cladosporium cladosporioides., Fusarium tricinctum., and Phomopsis helianthi..

Couladis et al. (2000) reported the essential oil of P. lanata. showed a moderate in vitro. activity against six Gram (+/−) bacteria and stronger activity against the three tested pathogenic fungi.

A new phenyethanoid glycoside, samioside, was isolated from the aerial parts of P. samia.; it demonstrated antimicrobial activity against Gram-positive and Gram-negative bacteria (Kyriakopoulou et al., 2001).

There is no report on the antimicrobial activity of the methanol extracts of Phlomis bruguieri., P. herba-venti., and P. olivieri.; therefore, we were unable to compare the antimicrobial activity of these plants.