Antimicrobial activity of Melampyrum cristatum , Melampyrum bihariense and Melampyrum arvense tinctures

The infections with bacteria and fungi resistant to existing chemotherapeutic drug have become a serious problem in the whole world, which leads to the continuous need to develop new antimicrobial agents. The plants extracts may represent future solutions against these pathogens. The purpose of this study was to determine the antibacterial and antifungal activity of Melampyrum cristatum, Melampyrum arvense and Melampyrum bihariense tinctures by using the diffusion and the dilution methods. The three tinctures inhibited the growth of Staphylococcus aureus up to a dilution of 1/8, and Escherichia coli up to a dilution of 1/4. The tinctures of M. cristatum and M. arvense inhibited the development of Pseudomonas aeruginosa up to a dilution of 1/4, while the tincture of M. bihariense up to a dilution of 1/8. Of the three tinctures, the tincture of M. cristatum showed the best antifungal action, inhibiting the development of Candida albicans up to a dilution of 1/8.


INTRODUCTION
The species of Melampyrum cristatum L., Melampyrum bihariense Kern., Melampyrum arvense are plants from the spontaneous flora of Romania, being part of the genus Melampyrum, family Scrophulariaceae.The name of Melampyrum comes from two Greek words: "melas"black and "pyros"-wheat, the seeds of this plant having the aspect of wheat kernels, but of black colour (Buia et al., 1960).The bacteria and fungi will continue to develop new mechanisms of resistance to chemotherapeutic drugs, which is why the plants extracts may represent future solutions against these pathogens.
The species of Melampyrum have been scarcely used in the traditional Romanian medicine (Butura, 1979).For M. arvense, the decoction of flower stalks was used in *Corresponding author.E-mail: anaionescuro@yahoo.com.
the Apuseni Mountains, in Ponor against hepatitis; the dried flower stalks were boiled with edible fruit (dried fruit) and the juice was drank.They were also used for baths against rheumatism.In Năsăud region, cattle with feet affections were washed with the plant decoction.M. bihariense it used sporadically for baths against rheumatism in combination with other plants in a tea against jaundice.M. cristatum decoction is held in the mouth for acute pain and infectious eruptions believed to have been picked up from found objects, were also washed with it.
The traditional use has been confirmed by the present studies regarding the diuretic and anti-inflammatory actions of M. bihariense and M. cristatum species (Mogoşan et al., 2008).
This research is the first performed in our country, studying the antimicrobial effects of the Melampyrum tinc- species, the presence of iridoid glycosides in this species as well as the antiprotozoal effect demonstrated in vitro over Trypanosoma brucei rhodesiense, Leishmania donovani and Plasmodium falciparum was highlighted by Munteanu et al. 2809 the gas chromatography-mass spectrometry (GC-MS) analysis (Kirmizibekmez et al., 2011).
As for the chemical composition of the Melampyrum genus species, the bibliographical data have shown the presence of several classes of compounds such as iridoid glycosides expressed in aucubin (Figure 4); flavonoids and carboxylic and polyphenolic acids (Figures 1  and 2) (Munteanu et al., 2008) expressed in luteolin and apigenin; sterols expressed in β-sitosterols (Figure 3), carotenoids and mucilages (Munteanu and Vlase, 2011).The analyses carried out to determine these classes of compounds were performed through the HPLC-MS technique.
The purpose of this study was to determine the antibacterial and antifungal activity of M. cristatum, M. arvense and M. bihariense tinctures.

MATERIALS AND METHODS
The species (M.bihariense and M. cristatum, were harvested in 2004 during the month of June; M. arvense was harvested during the month of May 2009) have been harvested from Mureş county, Romania, close to Lake Fărăgău, and analyses were carried out on the first species in 2004 and on the latter in 2009.
From the aerial parts of the species: M. cristatum, M. arvense and M. bihariense tinctures were prepared 1:10 (mg:ml) in ethylic alcohol of 70°C, according to FR X (Romanian Pharmacopeia, 1993).In order to test the antibacterial and antifungal activity, the following microorganisms have been used Staphylococcus aureus ATCC (American Type Culture Collection) 25933, Enterococcus faecalis ATCC 51299, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Proteus mirabilis ATCC 43071, Pseudomonas aeruginosa ATCC 27853, Bacillus cereus ATCC 11778, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404.Testing the antimicrobial activity was performed through the difusimetric method and the dilution method (in Mueller Hinton broth).The following substances were tested through the difusimetric method: tincture of M. cristatum (TMC), tincture of M. arvense (TMA), tincture of M. bihariense (TMB), and the ethylic alcohol.
Dilutions of 1/2, 1/4 and 1/8 were done using as diluent the alcohol in each tincture and 20 μl were applied in each well.The strains tested by the diffusion method were Gram positive bacteria: S. aureus ATCC 25933, Gram negative Bacteria: E. coli ATCC 25922 and P. aeruginosa ATCC 27853, and yeast: C. albicans ATCC 10231.The results were obtained in duplicate.
The three tinctures used were prepared as follows: from the aerial part of the three species, M. cristatum, M. arvense and M. bihariense tinctures were prepared 1:10 in 70° ethyl alcohol, according to FR X (Romanian Pharmacopeia, 1993).

Diffusion method
Diffusion method is also called the cup-plate agar diffusion method (Reeves and Reeves, 1983).The medium used was the simple Mueller-Hinton growth medium for screening S. aureus, E. coli, P. aeruginosa, and for C. albicans the medium used was Mueller-Hinton supplemented with 2% glucose (which provides a proper growth of yeasts) and 0.5 µg/ml methylene blue (provides a better delimitation of the diameter of the zone of inhibition).Six millimeter diameter wells were cut from the agar using a sterile cork-borer, and a predetermined (20 μl) volume of each compound solution were delivered into the wells.
For each of the tested microorganisms, a few colonies (maximum 5) were suspended from the pure culture in 5 ml of 0.85% sodium chloride (NaCl) sterile solution in order to obtain a suspension with standard turbidity of 0.5 McFarland at 530 nm (inoculum that corresponds to a population of 1×10 6 to 5×10 6 CFU).
After obtaining a suspension with standard turbidity, the medium was inoculated with a sterile nontoxic swab well impregnated in the prepared inoculum.The surface of the medium was inoculated in three directions in order to obtain a uniform distribution on the whole surface of the medium.After drying by maintaining at room temperature for 5 to 15 min, 20 μl from each tested substance was deposited in the well.
The plates incubated at 35°C for 20 to 24 h (Mareş, 2007), and the results were obtained in duplicate.

Dilution method
The dilution method had two parts: the first part was the simple dilution, which uses small glass beads, on the surface of which the microorganism to be tested was dried for 15 min.The beads were then exposed to the substance whose antimicrobial action was tested, at a temperature of 20°C, time of action at 1, 5, 10 and 30 min, and were rinsed with water, and then transferred into tubes with growth medium.After incubating at 37°C for 48 h, the tubes were examined for growth.

Germ carrier test
The strains tested through the simple dilution method with glass beads were S. aureus ATCC 25933, E. faecalis ATCC 51299, E. coli ATCC 25922, S. enteritidis ATCC 13076, P. mirabilis ATCC 43071, P. aeruginosa ATCC 27853, B. cereus ATCC 11778, C. albicans ATCC 10231, and A. niger ATCC 16404.The tested substances were undiluted TMC, TMA, and TMB.A suspension was prepared in 0.85% NaCl with a measured density of 10 7 CFU/ml for bacteria and of 10 7 to 10 8 CFU/ml for fungi for inoculum from 18 to 24 h culture.The microorganism's bearer is the glass beads.
The glass beads (bearer) were immersed into the bacterial suspension for 15 min, after which they were allowed to dry.The beads were introduced into the testing substance (or dilutions of this substance) and allowed to contact for different time periods.A culture witness was prepared with glass beads removed from the microbial suspension and which were not put in contact with the testing substance.Elimination of the antimicrobial substance was done by introducing the beads into sterile distilled water for a minute.For subcultures, the beads were each introduced into a tube with liquid medium: broth for bacteria and Sabouraud liquid medium for fungi.The tubes with liquid medium were incubated at 37°C for 18 to 72 h for bacteria and 7 days for fungi.The same procedure was used with culture control beads.The cultures have been microscopically examined and subcultures have been created on adequate mediums.In "high" means the development of the microorganism in the corresponding tube and in "absent" means lack of development of the microorganism in the corresponding tube and in subcultures.The second part of this method was the broth dilution method.
The inoculum was prepared from the pure culture of 18 to 24 h of the testing strain, adapted to the density of the standard of 0.5 McFarland.The inoculum thus standardized was diluted (1:10) in a sterile isotonic saline solution.The standardized culture was diluted as follows: 1/200 for Enterobacteriaceae and Staphylococci and 1/1000 for Pseudomonades, which ensured an inoculum of approximately 5×10 5 CFU/ml.Each tube received 0.1 inoculum from the binary dilutions from the antimicrobial substance in Mueller Hinton broth of 1 ml; the last tube was used as culture witness, and it contains 1 ml Mueller Hinton broth and 0.1 inoculum (it does not contain antimicrobial substance).The tubes were incubated at 37°C for 20 h and the smallest concentration was incubated as antimicrobial agent that inhibits development of the germ (lack of any macroscopically noticeable growth represents the minimum inhibitory concentration (MIC)) (Buiuc and Neguţ, 1999).By using broth dilution method, the tested strains were S. aureus ATCC 25933, E. coli ATCC 25922, P. aeruginosa ATCC 27853, C. albicans ATCC 10231.The tested substances were TMC, TMA, and TMB in various dilutions (Reeves and Reeves, 1983).

Diffusion method
After the incubation period, the area of complete inhibition (mm) was measured with the measuring rule in the light reflected against matte black background.The complete inhibition of the growth was followed.It was checked if the diameters of the zones of inhibition of each antimicrobial substance were within the limits of permissible variation (in accordance with the CLSI standard).The results obtained for S. aureus ATCC 25933 are shown in Table 1 (ethylic alcohol = 10 mm).The results obtained for E. coli ATCC 25922 are shown in Table 2 (ethylic alcohol = 10 mm).The results obtained for P. aeruginosa ATCC 27853 are shown in Table 3 (ethylic alcohol = 10 mm).The results obtained for C. albicans ATCC 10231 are shown in Table 4 (ethylic alcohol = 10 mm).
The results obtained following the simple dilution, with glass beads, the three analysed tinctures in undiluted form, after the contact time of 20 min, showed an antibacterial action (over Gram positive cocci: S. aureus, E. faecalis, Gram negative bacilli: E. coli, S. enteritidis, P. mirabilis, P. aeruginosa, and Gram positive bacilli: B. cereus) and an antifungal action (over the yeasts, C. albicans and filamentous fungi, A. niger).
The results obtained following Mueller-Hinton broth dilution method.The three tinctures analysed cause a growth inhibition up to the dilution of 1/8, as far as S. aureus is concerned.Regarding the behaviour of Melampirum species tinctures over E. coli, the data obtained were slightly different.Thus, for a dilution of ¼, the tinctures obtained from M. cristatum and M. bihariense showed a higher inhibition than in the case of M. arvense tincture.In the case of P. Aeruginosa, the best inhibition of the bacterial growth was obtained with M. cristatum tincture, for a dilution of 1/8.In the case of C. albicans, the M. arvense and M. bihariense tinctures inhibited the fungal growth in a dilution of 1/2, the M. cristatum tincture determined the growth inhibition to a dilution of up to 1/8.

Conclusions
According to our knowledge, this is the first research performed in our country, studying the antimicrobial effects of the Melampyrum tinctures.Following the tests, it was noticed that M. cristatum, M. bihariense and M. arvense tinctures have shown an antibacterial and antifungal action.All the three tinctures inhibited the development of S. aureus up to a dilution of 1/8, the development of E. coli up to a dilution of 1/4.The M. cristatum and M. arvense tinctures inhibited the development of P. aeruginosa up to a dilution of 1/4, and the M.
bihariense tincture up to a dilution of 1/8.Of the three tinctures, the M. cristatum tinctured showed the best antifungal action, inhibiting the development of C. albicans up to a dilution of 1/8.Thus, our study has shown that Melampyrum tinctures presented antibacterial and antifungal activity, which justifies further studies in this direction.

Table 1 .
Diffusion method results obtained for S. aureus.

Table 3 .
Diffusion method results obtained for P. aeruginosa.

Table 4 .
Diffusion method results obtained for C. albicans.