Epilobi Hirsuti Herba Extracts Influence the In Vitro Activity of Common Antibiotics on Standard Bacteria

Abstract Epilobium genus has been confirmed as an effective source of natural antimicrobials. However, the influence of Epilobi hirsuti herba derived products on usual antibiotics activity has not been studied. In this study, several standardized Epilobi hirsuti herba extracts (EHE) were evaluated in order to asses their potential effects on usual antibiotics tested on standard Gram-positive and Gram-negative bacterial strains in vitro. The results emphasized that the bacterial strains ranged from sensitive (MIC values between 50–200 μg GAE mL-1) (S. epidermidis ATCC 12228) to very resistant (E. coli strains), E. faecalis ATCC 29212 being practically immune to EHE. In terms of synergistic interaction, Tetracycline and Ampicillin combinations lead to the most important stimulatory effects, the diameters of the inhibition zone being even 60% bigger compared to the antibiotic alone. Synergistic effects between myricetin(galloyl) derivates and Tetracycline were also revealed on P. aeruginosa and E. coli strains. Together, it clearly demonstrated not only EHE’s own antimicrobial properties, but also their capacity to influence the antimicrobial potency of some common antibiotics. These results could be useful for the area of herbal medicines and as potential candidates in managing microbial resistance, but also for physicians and pharmacists using combined antibacterial therapy.

Regarding the antimicrobial activity of Epilobium genus, studies [ Similarly, studies [2] on flowers and leaves of Epilobium angustifolium L. collected from Croatia (Mt. Velebit) that were tested on standard ATCC and clinical isolated Staphylococcus aureus (including MRSA), Bacillus subtilis, Escherichia coli (including p-fimbriae positive strain), Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, C. tropicalis, C. dubliniensis and Saccharomyces cerevisiae confirmed antimicrobial potency of both plant pieces and MIC values ranging from 4.6 ± 0.2 to 18.2 ± 0.8 mg mL -1 . Our previous studies [14,17] on different whole and selective Epilobi hirsuti extracts using raw material (herba) from Romanian Carpathians highlighted the antimicrobial activity of the tested extracts against Staphylococcus aureus (standard ATTC and clinical isolated strains) and E. coli (standard ATTC strains).
Given the high therapeutic potential of Epilobium species, this work was aimed at evaluating potential interactions between some whole and selective, polar and non-polar, standardized Epilobi hirsuti herba extracts and several well known antibacterial drugs, and were tested on nine (standard) Gram-positive and Gram-negative bacteria.
This interest is motivated by current worldwide concern regarding the increasing phenomenon of bacterial resistance to antimicrobial drugs in parallel with more and more scientific proofs of the effectiveness of plant compounds in managing microbial infections. Therefore, the effect of cooperation between different plant compounds is well known, as well as that between plant compounds and chemical drugs leading to more complex biological benefits. The more so as there is ample literature data confirming additive and synergistic effects between plant compounds and susceptible antibiotics [18][19][20]
The filtrate obtained with ethanol (250 mL E8) was concentrated to dryness under reduced pressure using a rotary evaporator (Büchi) and the resulting sicc product was passed into 20% (v/v) propylene glycol (PG) solution to provide the exactly 5 mg total phenols (expressed as gallic acid equivalents [GAE]) per 1 mL of extract/sample. The resulting standardized propylene glycol extract is further called great willowherb whole extract (codified P8).
Other 250 mL of filtrate obtained with ethanol (E8) were concentrated under reduced pressure and the final sicc product was solved into 100 mL of distilled water. The resulting aqueous solution was further (manually) extracted, first with (3 × 100 mL) chloroform and then with (3 × 100 mL) ethyl acetate, 24 hours for each stage of extraction. Aqueous and ethyl acetate fractions were (separately) concentrated at residue and the residues were passed into 20% PG so as to obtain the identical final concentration of 5 mg total phenols (GAE) per 1 mL of sample; the chloroform fraction was also concentrated at spiss product then passed into 20% PG by the whole extract (P8) algorithm, thus obtaining the equivalent of the whole extract (P8) as concerning the total non-polar compounds. Three standardized fractions resulted: the aqueous fraction (codified P8aq), ethyl acetate fraction (codified P8ea) and chloroform fraction (codified P8chl), further called Epilobi hirsuti herba selective extracts.
The whole extract (P8) as well as the three selective extracts (P8aq, P8ea and P8chl) were separately divided into 2 mL Eppendorf tubes and stored at -8°C until microbiological studies.

Qualitative (HP)TLC analysis
Studies were performed according to Wagner and Bladt's [21] and Reich and Schibli's [22], general method for polyphenols assessment, and detailed in our previous studies [14,17].

Antimicrobial Activity Assay
Inoculum preparation: Nutrient broth (Oxoid CM0001) and Muller-Hinton agar (Oxoid CM0337) were used as culture media. Test microorganisms were stored in freezing conditions and activated by cultivation in Nutrient broth at 37°C for 24 hours. Overnight cultures were diluted with Mueller-Hinton broth until a turbidity equivalent to 0.5 McFarland standard was achieved (1−2 × 10 8 CFU mL -1 ) [24]. The purity of the cultures was verified by streaking each culture on Nutrient agar supplemented with 5% sheep blood.

Minimum inhibitory concentration (MIC) assay:
The experiments were conducted using the reference method for testing the in vitro activity of the antimicrobial agents [25] where MIC is the lowest concentration of an antimicrobial agent that, under defined in vitro conditions, prevents the appearance of visible growth of the microorganism after its incubation overnight [25,26]. The MIC assay was carried out using an accurate working scheme (Table 1) as follows: four dilution series were prepared, corresponding to the four vegetal extracts tested, the whole extract (P8) and the three selective extracts, aqueous (P8aq), ethyl acetate (P8ea) and chloroform (P8chl) fractions, each of them precisely quantified for total phenol content. The dilutions were made by incorporation of each vegetal extract into Muller-Hinton Agar (MHA); MHA + PG 20% (4.1 mL + 0.9 mL) were used as negative control. Total phenol concentrations in the resulting culture medium ranged in the 25-900 μg GAE mL -1 interval in the case of P8, P8aq and P8ea samples and between 1.25-45 μg GAE mL -1 in case of P8chl samples.
After the solidification of MHA containing vegetal extracts, the bacterial inoculums (10 4 CFU/spot) were placed on the medium surface [27] and the plates were incubated for 24 hours at 37°C. The results were interpreted by evaluating the bacterial growth, depending on the vegetal extract concentration. Synergism assay: In order to evaluate the potential synergistic effects between the four Epilobi hirsuti extracts and antibacterial substances tested, the vegetal extracts were incorporated in MHA in concentrations which did not by themselves inhibit the bacterial growth (MIC/2) [28]. Separately, plates with MHA without vegetal extract were prepared and used as controls. The dried surface of the culture medium was inoculated with bacterial suspension (1−2 × 10 8 UFC mL -1 ) by streaking with a sterile cotton swab and subsequently the antimicrobial micro-tablets were distributed. The plates were incubated for 24 hours at 37°C, and then the antimicrobial effect was evaluated by measuring the inhibition area around the micro-tablets.
Statistical analysis: All assays were conducted in triplicate and the values were expressed as mean ± (SD).
With regards to quantitative aspects, Epilobi hirsuti herba polar extracts including the whole extract (P8) as well as the two selective extracts, aqueous (P8 aq) and ethyl acetate (P8ea) fractions were made so as to assure identical total phenols content, 5 mg GAE mL -1 sample. As for the selective, non-polar, chloroform (P8chl) fraction, although chloroform is not a solvent in which polyphenols are typically soluble and extracted, our laboratory practice has revealed that chloroform extracts often contain small amounts of polyphenol compounds. In the particular case of Epilobi hirsuti herba extracts, the chloroform fraction (P8chl) showed 0.25 mg total phenols (GAE) mL -1 extract.
Therefore, the four Epilobi hirsuti herba extracts (EHE) differed in terms of qualitative content, but were identical in terms of quantitative content and the chloroform fraction was designed to provide the same amount of non-polar compounds as the whole extract does. This allows further assessments of Epilobi hirsuti herba polar and non-polar compounds' contribution to antimicrobial activity and synergistic effects in combination with usual antibiotics on standard Gram-positive and Gram-negative microbial strains.

Evaluation of the minimum inhibitory concentration (MIC)
Foremost it must be noted that during the tests, the nine bacterial strains properly developed on MHA and MHA + PG culture medium, thus demonstrating the lack of influence of propylene glycol solution (PG 20%, v/v) on bacterial growth. MIC values of the four Epilobi hirsuti herba extracts tested on the (nine) bacterial strains are shown in Table 2.
The results ( Table 2) indicated good antimicrobial activity of the three polar, polyphenolic extracts (whole/ P8, aqueous/P8aq and ethyl acetate/P8ea extracts), which were effective on five microbial strains, S. epidermidis ATCC 12228, P. mirabilis ATCC 29245, P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and S. aureus ATCC 6538 respectively; MIC values ranged between 50 -350 μg GAE mL -1 . Also, it clearly revealed the lack of activity of Epilobi hirsuti extracts on E. faecalis ATCC 29212 and the three E. coli strains (MIC > 500 μg GAE mL -1 ). The chloroform fraction (P8chl) proved to be effective mainly on S. epidermidis ATCC 12228 and, in a smaller extent, on S. aureus ATCC 25923. On the other seven microbial strains, the P8chl extract was basically ineffective.

Evaluation of the synergistic effects upon several usual antibiotics
Studies included six common antibiotics massively used for treating infections worldwide; their continuously increasing susceptibility to bacterial resistance is confirmed by official reports of the European Union [29]). Furthermore, each selected substance for the assay belongs to a separate class of antibacterials as follows: Penicillins (Ampicillin/AM), Aminoglycosides (Gentamicin/CN), Tetracyclines (Tetracycline/TE), Potentiated Sulphonamides (Sulfamethoxazole-Trimethoprim/SXT), Fluoroquinolones (Ciprofloxacin/ CIP), and Cephalosporins (Cefoxitine/FOX).
As the working principles, for those bacterial strains whose MIC values could not be determined (e.g., E. faecalis ATCC 29212) the synergism tests were not performed. For bacteria (e.g., S. epidermidis ATCC 12228) or fractions (e.g. P8chl) that showed MIC values lower than 200 μg GAE mL -1 (high-susceptible) or higher than 900 μg GAE mL -1 (low-susceptible), the synergism tests were not performed either; these samples are pointed out as ND (not determined). Significant synergy (stimulator effect) between the antimicrobial substances and Epilobi hirsuti herba extracts was considered when the enlargement of the combined inhibition zone of at least 5 mm was recorded [30]. The results are presented in Table 3.
Figs. 2-7 illustrate bacterial response after the treatment with respective vegetal-chemical antimicrobial combination (Epilobi hirsuti herba whole or selective extract plus antibiotic), stimulator response compared to the reference sample, relative ratio (%) respectively.
As can be seen in Fig. 2, the Tetracycline (TE) antibiotic acted much more effectively with whole and selective Epilobi hirsuti extracts on all tested strains, mainly on P. aeruginosa ATCC 27853 when combined with the ethyl acetate (P8ea) fraction previously shown as containning myricetin and myricetin-galloyl derivates; TE efficiency also increased on E. coli strains by combining with Epilobi hirsuti polar extracts, whole extract (P8), aqueous (P8eq) and ethyl acetate (P8ea) fractions respectively, as well as on S. aureus strains when combined with non-polar, chloroform (P8chl) fraction; TE and chloroform (P8chl) fraction also resulted in a increase of efficacy on P. mirabilis ATCC 29145.
Ampicillin (AM) antibiotic (Fig. 3) also acted more effective on two of the three E. coli strains when combined with Epilobi hirsuti polar extracts (P8, P8aq and Pea). Similar to TE, AM gained efficacy against S. aureus ATCC 25923 and P. mirabilis ATCC 29145, when combined with the non-polar, chloroform (P8chl) fraction.
Ciprofloxacin (CIP) indicated (Fig. 4) an increase of efficiency on S. aureus strains when combined with the chloroform (P8chl) fraction, as well as on P. aeruginosa *Note: Antimicrobial activity of the non-polar, P8chl fraction can be explained by particular non-polar compounds activity or by non-polar environment that favours antimicrobial activity of other compounds (e.g., polyphenols) thus allowing less augmented quantities of the latest. Given these and also considering the way in which the four extracts were prepared, in the specific case of chloroform fraction (P8chl), the above values (μg GAE mL -1 ) represent the effective concentration of polyphenols in P8chl at the same dilution series applied for the whole (P8), aqueous (P8aq) and ethyl acetate (P8ea) extracts and less MIC results.
ATCC 27853 by combining with all Epilobi hirsuti extracts, but especially with selective ethyl acetate (P8ea) fraction. Gentamicin (CN) in combination with tested extracts (Fig. 5)  Sulfamethoxazole/Trimethoprim (SXT) combinations with polar extracts (Fig. 6) indicated an increase of  efficiency on two of the three E. coli strains, mainly the aqueous (P8ea) fraction previously shown as containing two phenolics, chlorogenic acid and kaempferol glycoside. Cefoxitine (FOX) combinations (Fig. 7) lead to weak to moderate stimulatory effects on S. aureus and E. coli strains also by combining with non-polar and, respectively, polar extracts. SXT and FOX antibiotics, both of them gained efficacy against P. mirabilis ATCC 29245 by combining with non-polar, chloroform (P8chl) fraction.
In terms of the bacterial sensitivity (Figs. 8−14), for S. aureus ATCC 6538 (Fig. 8), the most significant synergistic effects were recorded for the combination of the non-polar, chloroform fraction (P8chl) with CIP, FOX, TE and SXT. Compared to reference sample (MHA + PG), there were obtained rises of inhibition zones measuring from 6 to 11 mm, meaning 19 to 33% stimulatory effects.
For S. aureus ATCC 25923 (Fig. 9), there were synergistic interactions for the combination of the nonpolar, chloroform (P8chl) fraction with TE, AM, FOX and CIP, the diameter of the inhibition zone increasing by 5 to 7 mm, quantified as 15 to 22% stimulatory effects.
P. aeruginosa ATCC 27853 (Fig. 10) indicated high sensitivity to TE and CIP combinations with all Epilobi hirsuti herba extracts, but especially with the selective, ethyl acetate (P8ea) fraction, leading to increases of the diameter of the inhibition zone by 22% (in case of CIP) and by 55% (in case of TE), compared to reference sample.
P. mirabilis ATCC 29245 (Fig. 11) showed an increase of sensitivity by combining TE, AM, SXT and FOX with  the Epilobi hirsuti non-polar fraction (P8chl) resulting in the increase of the inhibition zones from 17 to 26%, also compared to reference sample.
In case of the E. coli ATCC 35218 strain (Fig. 12), a synergistic activity between TE and two polar extracts, the whole extract (P8) and selective ethyl acetate (P8ea) fraction has been noticed; the diameter of the inhibition zone, antimicrobial effect respectively, increased by 20 to 36%.
E. coli ATCC 11229 strain (Fig. 13) significantly reacted when combined AM with Epilobi hirsuti polar extracts; the whole extract P8 (by 38%), the selective aqueous P8aq fraction (by 38%) and ethyl acetate P8ea (by 61%) fraction. Synergistic effects were also recorded by combining the aqueous P8aq fraction with TE (by 37%) and SXT (by 42%) antibiotics. E. coli ATCC 8739 strain (Fig. 14) reacted when TE was combined with Epilobi hirsuti herba polar extracts; the whole extract (P8) lead to the increase of the inhibition zone by 21%, the selective aqueous (P8aq) fraction by 33% and ethyl acetate (P8ea) fraction by 29%. Significant increases of the inhibition zone diameters were highlighted for the combination of the selective aqueous (P8aq) fraction and selective ethyl acetate (P8ea) fraction with SXT (by 28% and 21%, respectively) and FOX (by 20% for both fractions) as well as by combining Epilobi hirsuti herba whole extract (P8) with CIP (by 21%), all compared to reference sample.
Collectively, our results suggest synergistic effects between myricetin and/or myricetin-galloyl derivates and Tetracycline, on P. aeruginosa and E. coli strains. Similarly, an increased efficacy of Amoxicillin, Ampicillin and Cefoxitine in combination with myricetin has been previously reported [20].

Conclusions
Epilobim hirsutum L. is a valuable phytomedicine worldwide renowned for its benefits on skin and prostate tissues thus resulting in numerous plant derived products, herbal medicines, cosmetics and hygiene products as well. The most frequent commercial applications of Epilobium sp. refer to topical curative (anti-irritant, antiinflammatory and anti-oxidant) products. As for internal use, the extracts from Epilobium sp. can be found as different formulations (capsules, softgel capsules), single or in combination with other plant-derived products, primarily for prostate treatment. Romanian folk medicine and corresponding commercial products recommend Epilobi hirsuti plant derived products for treating urinary,  liver, gastric and intestinal disorders too. Given the high interest for Epilobi hirsuti plant derived products in managing urinary, but also various digestive system disorders, studying Epilobi hirsuti herba extracts capacity to influence the in vitro activity of some usual antibiotics is of high importance.
In this way, our studies indicated that, in terms of the susceptibility to Epilobi hirsuti herba extracts (EHE), the nine bacterial strains ranged from sensitive (S. epidermidis ATCC 12228) to very resistant (E. coli strains), E. faecalis ATCC 29212 being practically immune to Epilobium active compounds. Thus, if the polar extracts, including whole extract (P8), and aqueous (P8aq) and ethyl acetate (P8ea) fractions were very effective on S. epidermidis ATCC 12228, P. mirabilis ATCC 29245, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 strains (MIC values from 50 to 200 μg GAE mL -1 sample), the non-polar, chloroform (P8chl) fraction, was effective mainly on S. epidermidis ATCC 12228.
In terms of the synergistic interaction with some common antimicrobials (antibiotics), Tetracycline (TE) established the biggest number of interactions (eleven) and with all tested Epilobi hirsuti herba whole and selective extracts, this property being highlighted by the enlargement of the inhibition zones diameter by 19 to 55% compared to the antibiotic alone. Comparatively, Ciprofloxacin (CIP) established six synergistic interactions with all tested extracts with the exception of selective aqueous fractions, their intensity ranging from 14% to 33% compared to the antibiotic alone. Sulfamethoxazole/Trimethoprim (SXT) established six synergistic interactions with selective fractions only, their intensity ranging from 17% to 42%. Ampicillin (AM) established five synergistic interactions with all Epilobi hirsuti herba whole and selective extracts, their intensity ranging from 16% to 61%. Cefoxitine (FOX) established four synergistic interactions with selective fractions only, their intensity ranging from 16% to 31%, while Gentamicin (CN) revealed only weak stimulator effects in combination with both, polar and non-polar Epilobi hirsuti herba extracts.
Together, these results demonstrate not only Epilobi hirsuti herba extracts own antimicrobial properties, but also the capacity to influence the antimicrobial potency of some common antibiotics.
These results could be useful for the area of herbal medicines and as potential candidates in managing microbial resistance, but also for physicians and pharmacists using combined antibacterial therapy.