In vitro testing of antiviral, antibacterial, antifungal effects and cytotoxicity of selected Turkish Phlomis species

0239–3006/$ 20.00 © 2010 Akadémiai Kiadó, Budapest

Acta Alimentaria, Vol. 39 (2), pp. 119–125 (2010) DOI: 10.1556/AAlim.39.2010.2.2

IN VITRO TESTING OF ANTIVIRAL, ANTIBACTERIAL, ANTIFUNGAL EFFECTS AND CYTOTOXICITY OF SELECTED

TURKISH PHLOMIS SPECIES

B. Özcelika*, i. Orhanb, M. kartalc and B. kOnuklugilc

aDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University, 06330, Ankara. Turkey bDepartment of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara. Turkey

cDepartment of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06110, Ankara. Turkey

(Received: 11 June 2007; accepted: 17 June 2008)

The objective of this study was to examine antibacterial, antifungal and antiviral properties of selected Phlomis species (Lamiaceae) growing in Turkey. The petroleum ether and methanol extracts of the seven species, namely P. armeniaca Willd., P. bourgaei Boiss., P. leucophracta P.H. Davis & Hub.-Mor., P. lunariifolia Sibth. & Sm., P. lycia D. Don, P. pungens Willd. var. pungens, and P. pungens var. hirta Velen. were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis for their antibacterial activity using ampicillin and oflaxocin as references. Antifungal activity of the same extracts was determined against Candida albicans using microdilution method with ketocanazole as reference. Both DNA virus Herpes simplex type-1 (HSV-1) and RNA virus Parainfluenza (PI-3) were employed for antiviral assessment of the Phlomis extracts using Madin-Darby Bovine Kidney and Vero cell lines in which acyclovir for HSV-1 and oseltamivir for PI-3 were employed as reference drugs. Although both the petroleum ether and methanol extracts seemed to exert similar antibacterial activity, the methanolic extracts were observed to be more active against S. aureus and E. faecalis. On the other hand, methanolic extract of P. pungens var. pungens possessed notable antiviral activity against both type of viruses.

Keywords: Phlomis, Lamiaceae, antiviral, antimicrobial, cytotoxicity

Microbial activity is a primary mode of deterioration in many foods. Currently, an increasing tendency is being observed to use natural antibacterial agents such as some plant extracts, herbs and spices in order to preserve foods. Plants can produce antimicrobial compounds to protect themselves from biotic attack that could be essential for microbial infection resistance. Thus, efforts have been made to discover new antimicrobial agents and plants have been reported to be quite preferable in this search (Vlietinck & Van Den Berghe, 1991).

The Phlomis genus belonging to Lamiaceae family consists of thirty-four species and ten hybrids in the Turkish flora (HuBer-MOrath, 1982). In Turkish folk medicine, a number of Phlomis species have been reported to be used as stimulant and tonic (BaytOp, 1999). Numerous phytochemical studies have been performed on different Phlomis species up to now and essential oil analysis, flavonoids, iridoids, phenylethanoids, lignans and their glycosides have been widely described (Zhang et al., 1991; TOMas-BarBeran et al., 1992; Çaliş, et al., 2005).

Various industries are looking into new natural sources of environmentally friendly antimicrobials and food protection agents. It is well known that Lamiaceae species have been

* To whom correspondence should be addressed.Phone: +90-312-2023261; fax: +90-312-2235018; e-mails: [email protected], [email protected].

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also used all over the world for seasoning, perfumes, and/or phytotherapeutical purposes. For this purpose, the petroleum ether and methanolic extracts of seven Phlomis species growing in Turkey were tested against selected strains of bacteria for their antibacterial activity, against yeast Candida albicans for their antifungal activity and against DNA virus Herpes simplex type-1 (HSV-1) and RNA virus Parainfluenza (PI-3) for their antiviral activity.

1. Materials and methods

1.1. Plant materials

Collection sites and herbarium numbers of the respective Turkish Phlomis species (P. armeniaca Willd., P. bourgaei Boiss., P. leucophracta P.H. Davis & Hub.-Mor., P. lunariifolia Sibth. & Sm., P. lycia D. Don, P. pungens Willd. var. pungens, and P. pungens var. hirta Velen.) in this study are given as follows: P. armeniaca (AEF 22974): Sandikli town-Afyon province, P. bourgaei (AEF 22979) and P. lycia (AEF 22980): Korkuteli town-Antalya province, P. leucophracta (AEF 22981) and P. lunariifolia (AEF 22971): Mahmutlar village of Alanya-Antalya province, P. pungens var. pungens (AEF 22984a) and P. pungens var. hirta (AEF 22984b): Beyşehir town-Konya province. All plant materials were collected in June 2002 and identified by Prof. Dr. Hayri Duman of the Department of Biology, Faculty of Art and Science, Gazi University, Ankara (Turkey). Voucher specimens are preserved at the Herbarium of Faculty of Pharmacy of Ankara University, Ankara (Turkey).

1.2. Preparation of the extracts

Air-dried and powdered plant materials were accurately weighed and macerated firstly with petroleum ether (Merck) and sequentially with methanol (Merck). After filtration, the relevant organic phases were combined, concentrated under vacuum to dryness and the crude petroleum ether (PE) and methanol (MeOH) extracts were obtained and the yields (w/w) are given, respectively, for PE and MeOH extracts as follows: P. armeniaca-PE and MeOH (5.4 and 63.2%), P. bourgaei-PE and MeOH (6.8 and 54.6%), P. leucophracta-PE and MeOH (5.8 and 47.2%), P. lunariifolia-PE and MeOH (7.5 and 35.0%), P. lycia-PE and MeOH (6.8 and 81.9%), P. pungens var. pungens-PE and MeOH (6.5 and 94.2%), P. pungens var. hirta-PE and MeOH (8.5 and 54.3%).

1.3. Preparation of the tested samples

The extracts were dissolved in ethanol:hexane (1:1) by using 1% tween 80 solution at a final concentration of 1024 µg ml–1 and sterilized by filtration using 0.22 µm Millipore (MA 01730, USA) and was used as the stock solutions. Standard antibacterial powders of ampicillin (AMP; Fako), ofloxacin (OFX; Hoechst Marion Roussel), standard antifungal powders of ketoconazole (KET; Bilim) were obtained from their respective manufacturers and dissolved in phosphate buffer solution (ampicillin; pH: 8.0; 0.1 mol l–1), dimethylsulphoxide (ketoconazole) and in water (fluconazole, ofloxocin). The stock solutions of the agents were prepared in a medium according to the National Committee for Clinical Laboratory Standards (NCCLS, 2002).

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1.4. Microorganisms

Standard strains of the following bacteria Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 10145, Proteus mirabilis ATCC 7002, Klebsiella pneumoniae RSKK 574, Acinetobacter baumannii RSKK 02026, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and Enterococcus faecalis ATCC 29212 were used for the determination of antibacterial activity and Candida albicans ATCC 10231 for the determination of antifungal activity.

1.5. Inoculum preparation

Mueller-Hinton Broth (MHB; Oxoid) and Mueller-Hinton Agar (MHA; Oxoid) were applied for growing and diluting the bacteria. Sabouraud liquid medium (SLM; Oxoid) and Sabouraud dextrose agar (SDA; Oxoid) were applied for growing and diluting the yeast. The medium RPMI-1640 with L-glutamine was buffered at pH 7 with 3-[N-morpholino]-propansulfonic acid (MOPS). Prior to the test, yeast and bacteria strains were cultured on media and passaged at least twice to ensure purity and viability at 35 oC for 24 to 48 h. The bacterial suspensions of 105 CFU ml–1 were prepared by diluting fresh cultures at McFarland 0.5 density (108 CFU ml–1). The fungal suspension was prepared by the spectrophotometric method of inoculum preparation at a final culture suspension of 2.5×103 CFU ml–1 (Özçelik et al., 2006).

1.6. Antibacterial and antifungal tests

The microdilution method was employed for antibacterial and antifungal activity tests. After placing media into each well of the 96 well plates, the extract solutions at 1024 µg ml–1 were added into the first raw of microplates and twofold dilutions of the compounds were made by dispensing the solutions to the remaining wells to give 12 concentrations in the range 512–0.25 µg ml–1 for each extract. Ten μl of culture suspensions were inoculated into all the wells. The sealed microplates were incubated at 35 ºC for 24 h and 48 h in a humid chamber. The lowest concentrations of the extracts that completely inhibit macroscopic growth and minimum inhibitory concentrations (MICs) were determined. Antibacterial activity of the extracts was tested against three Gram-positive, five Gram-negative bacterial strains, using ampicillin and ofloxacin as references. The yeast-like fungus, C. albicans, was included in the array of antifungal screens with the reference ketoconazole (Özçelik et al., 2008).

1.7. Cell line and growth conditions

Vero cell line (African green monkey kidney) and Madin-Darby bovine kidney (MDBK) used in this study were obtained from the Department of Virology, Faculty of Veterinary, Ankara University (Turkey). The cell cultures were grown in Eagle’s Minimal Essential Medium (EMEM) enriched with 10% fetal calf serum (FCS; Biochrom, Germany), 100 mg ml–1 streptomycin and 100 IU ml–1 penicillin in a humidified atmosphere of 5% CO2 at 37 ºC, and harvested using trypsin solution (Bipco Life Technologies, UK).

1.8. Test viruses

In order to determine the antiviral activity of the extracts, Herpes simplex virus type-1 (HSV-1) and Parainfluenza-3 virus (PI-3) were employed.

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1.9. Antiviral activity

Following the placement of media (EMEM) into each well of the 96-well microplates (GreinerR, Germany), stock solutions of the extracts were added into the first raw of microplates and twofold dilutions of the extracts (512–0.25 µg ml–1) were made by dispensing the solutions into the remaining wells. Twofold dilutions of each material were obtained according to Log2 on the microplates. Acyclovir (Biofarma) and oseltamivir (Roche) were used as references. Strains of HSV-1 and PI-3 titers were calculated as TCID50 and inoculated into all the wells. The sealed microplates were incubated in 5% CO2 at 37 °C for 2 h to detect the possible antiviral activities of the samples. After incubation, 50 µl of the cell suspension of 300,000 cells ml–1, which were prepared in EMEM together with 5% fetal bovine serum, were put in each well and the plates were incubated in 5% CO2 at 37 °C for 48 h. After the end of this time, the cells were evaluated using cell culture microscope (× 400) comparing with treated–untreated control cultures and acyclovir and oseltamivir. Consequently, maximum cytopathogenic effect (CPE) concentrations as the indicator of antiviral activities of the extracts were determined (Özçelik et al., 2009).

1.10. Cytotoxicity

The maximum non-toxic concentration (MNTCs) of each sample was determined by the method described previously by Özçelik and co-workers (2006) based on cellular morphologic alteration. Several concentrations of each sample were placed in contact with confluent cell monolayer and incubated in 5% CO2 at 37 °C for 48 h. MNTCs were determined by comparing treated and controlling untreated cultures (Özçelik et al., 2009).

2. Results and discussion

Results of comparative antibacterial and antifungal activities of the petroleum and methanol extracts of seven Phlomis species growing in Turkey were tested against selected bacteria and C. albicans are shown in Table 1. No difference was observed between antibacterial and antifungal efficiencies of the extracts, except that antibacterial activity was slightly higher in the methanol extracts against S. aureus, B. subtilis, and E. faecalis. In particular, it is notable to state that MIC values (2 µg ml–1) of the methanol extracts for E. faecalis were highly comparable to that of ofloxacine (1 µg ml–1).

According to our results obtained from antiviral tests in this survey, the petroleum ether extracts were not active towards PI-3 and only P. armeniaca and P. bourgaei were found to be moderately effective against HSV-1, while they were less cytotoxic than acyclovir and oseltamivir (64 µg ml–1). The methanol extract of P. pungens var. pungens was the only active one against PI-3 at the concentration of 16–32 µg ml–1. Additionally, the same extract along with P. lycia displayed activity against HSV-1. Moreover, P. pungens var. pungens had nearly the same MNTC value (32 µg ml–1) as acyclovir and oseltamivir.

Up to date, there have been more than a number of antimicrobial studies on various Phlomis species. In a similar study on antibacterial activity of the chloroform extract of P. bourgaei of Turkish origin, by determining the inhibition zones the extract was reported to be moderately effective against B. brevis, B. subtilis, B. cereus, P. aeruginosa, S. aureus, K. pneumoniae, P. vulgaris, Micrococcus luteus, and Mycobacterium smegmatus, whereas it was completely inactive against C. albicans (Digrak et al., 1999). Interestingly, our results for P. bourgaei seemed not to overlap with the aforementioned data, which might depend

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on either the localities where they were collected or on the methods used for determination of antibacterial activity. COuladis and co-workers (2000) reported that the essential oil of P. lanata growing in Greece showed a moderate antibacterial activity against S. aureus, S. epidermidis, P. aeruginosa, E. coli, and E. cloacae, as well as a strong antifungal activity against C. albicans. α-Pinene was the major component of the essential oil of this plant, which was suggested to be responsible for its antimicrobial potential. Ristic and co-workers (2000) also studied the antimicrobial activity of the essential oil and ethanol extract of P. frutica and found that the essential oil had inhibitory effect towards S. aureus, E. coli, B. subtilis, K. pneumoniae, and Micrococcus luteus, as well as towards the fungi Aspergillus niger, A. ochraceus, Cladosporium cladosporioides, Fusarium tricinctum, and Phomopsis helianthi, while the ethanol extract was active only against S. aureus and B. subtilis along with the fungi A. niger, A. ochraceus, C. cladosporioides, F. tricinctum, and P. helianthi. In another study, antiviral activity of the ethanol and aqueous extracts of P. lychnitis and L. herba-venti growing in Spain was assessed against HSV-type 1, vesicular stomatitis virus (VSV), and poliovirus-type 1 and was found to be inactive (ABad et al., 2000). In a study of

Table 1. Antibacterial and antifungal activity of the extracts and the references as Minimum Inhibitory Concentrations (MICs; µg ml–1)

Microorganisms Plants studied PE Extracts E.

col

i

P. a

erug

inos

a

P. m

irab

ilis

K. p

neum

onia

e

A. b

aum

onni

i

S. a

ureu

s

B. su

btili

s

Ent.

faec

alis

C. a

lbic

ans

P. armeniaca 128 128 128 64 64 8 128 4 8

P. bourgaei 128 128 128 64 64 8 128 4 8

P. leucophracta 128 128 128 64 64 8 128 4 8

P. lunariifolia 128 128 128 64 64 8 128 4 8

P. lycia 128 128 128 64 64 8 128 4 8

P. pungens var. pungens 128 128 128 64 64 8 128 4 8

P. pungens var. hirta 128 128 128 64 64 8 128 4 8

MeOH extracts

P. armeniaca 128 128 128 64 64 4 64 2 8

P. bourgaei 128 128 128 64 64 4 64 2 8

P. leucophracta 128 128 128 64 64 4 64 2 8

P. lunariifolia 128 128 128 64 64 4 64 2 8

P. lycia 128 128 128 64 64 4 64 2 8

P. pungens var. pungens 128 128 128 64 64 4 64 2 8

P. pungens var. hirta 128 128 128 64 64 4 64 2 8

References

AMP 2 – 2 2 2 <0.12 0.5 0.5 –

OFX 0.12 1 <0.12 0.12 0.12 0.5 1 1 –

KET – – – – – – – – 1 AMP: Ampicilline; OFX: ofloxasine; KET: ketoconazole; –: not tested

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BedOya and co-workers (2002), the aqueous extracts of the above-mentioned two Phlomis species were examined for their ability to inhibit human immunodeficiency virus (HIV) replication. However, no anti-HIV effect was shown by those extracts, and no toxicity was observed either with P. herba-venti at all even at the highest concentration (400 µg ml–1). P. kurdica collected from Turkey did not possess anti-HSV and anti-HIV capacity against neither types I and II (SÖkMen, 2001). In another previous study, the phenylethanoid glycosides (verbascoside, isoacteoside, decafeoylacteoside, martynoside, leucosceptosides A and B, β-hydroxyacteoside, forsythoside B, alyssonoside, myricoside, and samioside) isolated from P. viscosa of Turkish origin were tested against E. coli, P. aeruginosa, S. aureus, E. faecalis, as well as against the fungi C. albicans, C. parapsilosis, and C. krusei (Çaliş et al., 2005). All of these compounds were reported to be inactive towards Gram-negative bacteria and

Table 2. Antiviral activity of the extracts and the references

Plants studied

MDBK cells(µg ml–1)

Vero cells(µg ml–1)

MNTC(µg ml–1)

CPE inhibitory concentrationMNTC

(µg ml–1)

CPE inhibitory concentration

HSV-1 PI-3

Max. Min. Max. Min.

PE extracts

P. armeniaca 64 32 16 64 – –

P. bourgaei 64 32 16 64 – –

P. leucophracta 64 – – 32 – –

P. lunariifolia 64 – – 64 – –

P. lycia 64 – – 32 – –

P. pungens var. pungens

64 – – 32 – –

P. pungensvar. hirta

64 – – 32 – –

MeOH extracts

P. armeniaca 128 – – 32 – –

P. bourgaei 16 – – 8 – –

P. leucophracta 8 – – 4 – –

P. lunariifolia 16 – – 8 – –

P. lycia 8 8 4 4 – –

P. pungens var. pungens

32 16 8 32 32 16

P. pungensvar. hirta

32 – – 2 – –

References

Acyclovir 16 16 <0.25 – – –

Oseltamivir – – – 32 32 <0.25

MNTC: Maximum non-toxic concentration; CPE: cytopathogenic effect, –: no activity observed

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C. albicans, whereas verbascoside, isoacteoside, forsythoside B, samioside, leucosceptoside A and myricoside inhibited Gram-positive bacteria weakly.

3. Conclusion

The phenylpropanoid and iridoid glycosides, widely distributed in Phlomis species, have also been reported to exhibit antimicrobial and cytotoxic effects (Pan et al., 2003). Although most of the Phlomis species studied herein did not sparkle very much for their antimicrobial effects, it is still worth to evaluate anti-Enterococcus and antifungal effects of these species as well as antiviral activity of P. pungens var. pungens from the viewpoint of chemical identification of active constituents. To best of our knowledge, this is the first report describing antimicrobial activities of P. leucophracta, P. lunariifolia, P. lycia, P. pungens var. pungens, and P. pungens var. hirta.

*

We would like to thank Dr. T. KaraOglu for technical assistance in this study.

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