ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM IN VIVO AND IN VITRO PROPAGATED LAMIUM ALBUM L. PLANTS

The antimicrobial activity of 18 different extracts from in vivo and in vitro grown L. album L. plants was evaluated against clinical bacteria and yeasts using the well diffusion method. All the used extracts demonstrated antibacterial activity, whereas only the water extracts from leaves ( in vivo ) possessed antifungal activity against Candida albicans NBIMCC 72 and Candida glabrata NBIMCC 8673 (14 and 20 mm diameter of inhibition zones and MIC 10 mg/ml, respectively). The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those from in vivo plants, while the opposite tendency was observed for the chloroform extracts. All tested flower extracts possessed antimicrobial activity. The chloroform extract from in vivo flowers demonstrated the highest activity against E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339 and P. aeruginosa NBIMCC 3700 (22 mm, 13 mm, 11 mm, 23 mm zone diameter of inhibition and MIC 0.313 mg/ml, respectively). The water extracts from leaves (both in vivo and in vitro ) possessed higher antibacterial activity than extract from flowers. The obtained results showed that both in vivo and in vitro propagated L. album L. could be used as a source of antibacterial substances. The present study revealed that all the tested L. album extracts possessed antimicrobial activity that varied depending on the test-microorganisms, solvents, the method of extraction, and the parts of the plants used in both in vivo and in vitro variants. The Gram-positive bacteria were more sensitive than the Gram-negative bacteria. The methanol and the ethanol leaf extracts obtained by the in vitro propagated L. album L. possessed a broader spectrum of antibacterial activity than the in vivo extracts. The lowest MIC was demonstrated by the chloroform extracts (from in vivo flowers and leaves) towards E. faecalis , S. aureus , P. hauseri , and P. aeruginosa . The obtained results showed that L. album L. could be of interest for biotechnology since it can be used as a natural source of antimicrobial substances as an alternative to chemical therapeutics.

Since there was no available data about the cultivation of L. album L. in vitro, the aim of this study was to evaluate the antibacterial activity of wild L. album plants cultivated in both, in vivo and in vitro conditions.

Plant material
Above-ground material was collected from mature L. album plants harvested in the Lozen Mountain, near Sofia, Bulgaria.The voucher specimen 105183 has been deposited in the Herbarium of the Department of Botany, Faculty of Biology, Sofia University, Sofia, Bulgaria.In vitro cultures were induced from sterilized mono-nodal stem segments of the mature wild plants.The plants were sterilized and propagated under controlled environmental conditions in vitro (Dimitrova et al., 2010), and after four weeks of cultivation they were collected and air-dried.For the in vivo variants (leaves and flower), the mature plant material was harvested from the natural habitats and dried in the shade for grinding.

Plant extracts Soxhlet extraction
Samples of 3 g from in vivo (leaves and flower) and in vitro powdered air-dried L. album plants were used.The material was subjected to Soxhlet extraction by chloroform and methanol as solvents.Afterwards, the solvents were removed by a rotary evaporator, and the extracts were concentrated, dried, and kept in dark at 4 o C.

Thermostat extraction
Samples of 2 g from in vivo (leaves and flower) and in vitro air-dried L. album plants were extracted according to the method described by Schinella et al. (2002) for 24 h at 40 o C by the following solvents -chloroform, ethanol, methanol, and water.After filtration the raw materials were extracted twice in the same conditions.The solvents were removed under a vacuum evaporator, and the extracts were concentrated, dried, and kept in dark at 4 o C.

Antimicrobial activity
The antimicrobial activity was determined by the well diffusion method according to National Committee for Clinical Laboratory Standards (NCCLS -1997).Petri plates with 20 ml of Muller Hinton Agar medium (MNB, Difco Laboratories, Detroit, MI, USA) for bacteria and Potato Dextrose Agar (Oxoid) for yeasts were inoculated with 1.5.10 7 cfu/ml (0.5 McFarland standards) of 24 h bacterial or yeast suspensions.Wells were cut into agar and filled with 50 μl (10 mg/ml) of plant extracts.Standard disks (Oxoid) impregnated with tetracycline (30 μg/disk) and nystatine (100 IU/disk) were used as positive controls.Sterilized paper disks impregnated with pure solvents (10 μg/disk) served as negative controls.The plates were placed for 2 h at 4 o C to allow the diffusion of extracts and cultivated for 24 h at 37 o C. The antimicrobial activity was assessed by measuring the diameter of the inhibition zone around the well.All experiments were performed in triplicate.

Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was determined by the micro-dilution method using paper disks.Each extract was diluted twofolds and used for preparation of serial dilutions with a final concentration from 10 mg/ml to 0.078 mg/ml.Sterile paper disks were impregnated with 50 μl of the diluted extracts, dried and placed onto agar medium which was previously inoculated with test-microorganisms.After diffusion of the extract for 2 h at 4 o C the plates were cultivated for 24 h at 37 o C. The antimicrobial activity was assessed by measuring the diameter of the inhibition zone around the disks.All experiments were performed in triplicate.The lowest concentration of the extract that formed inhibition zone was regarded as MIC.

Results and Discussion
Eighteen different L. album extracts were screened for antimicrobial activity (Table1).The extracts were obtained from leaves and flowers of mature L. album plants which were harvested either in their natural habitats, or from in vitro propagated plants.Four solvents (chloroform, methanol, ethanol, and water) and two methods of extraction (Sоxhlet, thermostat) were used.The antimicrobial activity of screened plant extracts is shown in Table 1.All the extracts obtained by the different solvents exhibited antibacterial activity, and only the water extracts (extracts 10 and 13) possessed antifungal activity.The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those obtained from the in vivo plants.The opposite tendency was observed for the chloroform extracts (Table 1).The chloroform extracts obtained by the Soxhlet method and the methanol extracts collected by the thermostat method showed higher activity compared to the others.All flower extracts possessed antimicrobial activity but the chloroform extract obtained by the Sоxhlet method had the broadest spectrum by inhibiting ten from the eleven tested Gram-positive and Gramnegative bacteria.The activity varied among the different strains with highest sensitivity observed for E. faecalis 3915 and P. aeruginosa 3700 (Table 1).All bacterial and yeast strains were sensitive to the respective antibiotic controls.Pure solvents did not possess inhibitory effect.
The wide spectrum of antibacterial activity of L. album plant extracts might be related to the presence of some biologically active substances.Indeed, the medicinal properties of this plant had attracted significant attention and intensive phytochemical investigations had been done.Among the secondary metabolites of L. album L., terpenoids (Brieskorn and Ahlborn, 1973;Eigtved et al., 1974;Damtoft, 1992;Sarker et al., 1997), phenylethanoids (Budzianowski and Skrzypczak, 1995), and phytoecdysteroids (Savchenko et al., 2001) had been reported.In a Danish population of L. album L. four iridoids and secoiridoids were isolated (Eigtved et al., 1974;Damtoft, 1992) whereas in a Bulgarian population several flower volatile compounds, iridoid glucosides (Alipieva, 2003;Alipieva et al., 2006), as well as phenols and flavonoids (Valyova et al., 2011) were detected.Phenols and flavonoids were found in both in vivo and in vitro propagated L. album plants (Valyova et al., 2011).Up to date, only the essential oil of L. garganicum subsp.laevigatum was reported to be active against some Gram-positive and Gram-negative bacteria (Roussis et al., 1996).Based on the published data, it could be assumed that in our study the antimicrobial activity of the screened L. album extracts is due to the presence of such biologically active compounds.The MIC was determined for the extracts with higher activity (Table 2).The MIC values varied among the extracts and the microbial strains, and ranged from 10 to 0.313 mg/ml.The lowest MIC (0.313 mg/ml) was observed as follows: in chloroform extract from flowers (by the Soxhlet method) towards E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339, P. aeruginosa NBIMCC 3700; in ethanol extract from leaves (in vitro) towards E. aerogenes NBIMCC 3699; and in chloroform extracts from leaves and flowers (by the thermostat method) towards P. aeruginosa NBIMCC 3700.

Conclusion
The present study revealed that all the tested L. album extracts possessed antimicrobial activity that varied depending on the testmicroorganisms, solvents, the method of extraction, and the parts of the plants used in both in vivo and in vitro variants.The Gram-positive bacteria were more sensitive than the Gram-negative bacteria.The methanol and the ethanol leaf extracts obtained by the in vitro propagated L. album L. possessed a broader spectrum of antibacterial activity than the in vivo extracts.The lowest MIC was demonstrated by the chloroform extracts (from in vivo flowers and leaves) towards E. faecalis, S. aureus, P. hauseri, and P. aeruginosa.The obtained results showed that L. album L. could be of interest for biotechnology since it can be used as a natural source of antimicrobial substances as an alternative to chemical therapeutics.

Table 1 .
Antimicrobial activity of the crude extracts of L. album L. to the test-microorganisms

Table 2 :
MIC of different extracts of L. album L. on the test-microorganisms