Burdock (Arctium Lappa) Leaf Extracts Increase the in Vitro Antimicrobial Efficacy of Common Antibiotics on Gram-Positive and Gram-Negative Bacteria

Open Chem., 2017; 15: 92–102

Research Article Open Access

Lucia Pirvu*, Isabela Nicorescu, Cristina Hlevca, Bujor Albu, Valentin Nicorescu Burdock (Arctium lappa) Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Common Antibiotics on Gram-positive and Gram-negative Bacteria

DOI 10.1515/chem-2017-0012 received January 23, 2017; accepted March 14, 2017. inhibitory) of Arctii folium extracts in combination with typical antibiotics as well as a potential use of the whole Abstract: This work aimed to study the potential effects of ethanol extract/W for restoring the antimicrobial potency four Arctii folium extracts, 5 mg gallic [GAE] acid equivalents of susceptible antibiotics have also been evidenced. per 1 mL sample, on six antibiotics (Ampicillin/AM, Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole- Keywords: burdock leaves, interaction with usual Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/ antibiotics, stimulatory and inhibitory effects CN) tested on four Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Staphylococcus 1 Introduction epidermidis ATCC 12228) and five Gram-negative (Proteus mirabilis ATCC 29245, Escherichia coli ATCC 35218, E. coli Arctium lappa L. (Asteraceae family), commonly greater ATCC 11229, E. coli ATCC 8739, and Bacillus cereus ATCC burdock, is a biennial species found across most of tEurope, 11778) bacteria. Arctii folium extracts were the whole Asia and also America. The root part, Bardanae radix, is ethanol extract/W and subsequent ethyl acetate/EA, used as a herbal medicine, but is also used as a vegetable aqueous/AQ, and chloroform/CHL fractions. Chemical in Eastern Asian regions (similar to potatoes) due to its qualitative analysis (HPTLC method) emphasized five nutritional value [1], the high content of carbohydrates main polyphenol compounds in Arctii folium polar (69%) including inulin (27-50%) and mucilage added to extracts: chlorogenic acid (Rf≈0.52/0.55) and its isomer, fats, proteins and numerous vitamins (B1, B2, B3, B5, B6, 1,5-di-O-caffeoylquinic acid (Rf≈0.90/0.92), plus cynarin B9, C, E and K) and minerals (Ca, Fe, Mg, Mn, P, K, Na and (Rf≈0.77), hyperoside (Rf≈0.68/0.64) and isoquercitrin Zn) [2] respectively. (Rf≈0.69/0.71). Microbiological screening indicated Arctii Concerning the basis of its medicinal use, data folium polar extracts (AQ and W) efficacy on S. epidermidis indicate that Bardanae radix is rich in caffeoylquinic acid ATCC 12228; the MIC values were in the range of common isomers [1, 3], with dicaffeoylquinic acid derivatives being antibiotics, being 32 and 128 μg GAE per mL sample quantified at 75.4% and 1,5-di-O-caffeoyl-4-O-maloylquinic respectively. The unpredictable effects (stimulatory or acid representing 44% of the total isomers [4], but that the content also comprisesnumerous other secondary metabolites such as tannin-iron complexes, various polyacetylenes and sulphuric actylene compounds, *Corresponding author: Lucia Pirvu: National Institute of Chemical- Pharmaceutical Research and Development (ICCF), Vitan 112, essential oils, guainolides and bitter compounds, lignans 031299,Bucharest, Romania, E-mail: [email protected] (e.g., arctigenin, arctiin,) and sterols (e.g., sitosterol, Isabela Nicorescu: Institute of Hygiene and Veterinary Public Health, stigmasterol) [2]. Studies regarding the metabolic profile Campul Mosilor 5, 021201, Bucharest, Romania of the compounds of burdock plant pieces indicated Cristina Hlevca, Bujor Albu: National Institute of Chemical- caffeoylquinic acid derivates and luteolin and quercetin Pharmaceutical Research and Development (ICCF), Vitan 112, rhamnosides occur in the root part, caffeoylquinic acid and 031299,Bucharest, Romania Valentin Nicorescu: University of Agronomic Sciences and Veterinary quercetin, quercitrin and luteolin derivates in the leaves, Medicine, Faculty of Veterinary Medicine, Splaiul Independentei whereas cynarin and chlorogenic acid are dominant in the 105, 050097, Bucharest, Romania seeds [5].

© 2017 Lucia Pirvu et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 93

Based on this valuable phytochemical content, growing in Romania, this study was aimed to evaluate any Bardanae radix represents an important ingredient potential interaction of Arctii folium extracts with typical for traditional medicine practitioners, and has also antibiotics. The results of the study are very useful to avoid achieved international recognition for its numerous situations of incompatibility with such antibiotics, but also healthy effects [1]; Romanian folk medicine also considers as the starting point for developing new natural synergistic a bardanae radix hot water extract (decoction type) products able to restore antimicrobic susceptibility. as an effective expectorant, anti-tussive, emollient, diuretic and anti-inflammatory remedy thus being recommended for different lung, digestive, renal and skin ailments [6]. Proving these, studies have reported 2 Experimental Procedure burdock products and corresponding separate extracts or specific compounds (especially lignans such as arctiin 2.1 Materials and arctigenin, and polyphenols compounds) having antioxidant, anti-inflammatory, antitumor, antibacterial 2.1.1 Plant material description and antiviral activities [1], as well as hepatoprotective [7], antiurolithiasic [8] and skin improving [1, 9] effects, with Leaves from burdock (Arctii folium) were harvested in some clinical studies also proving their effectiveness in June and September from the Romanian Carpathians, knee osteoarthritis [10] and for dental infectious conditions Sinaia region (1,000 m altitude). Taxonomic identification [11, 12]; burdock root and fruits are also reported to have was carried out by the botanist’s team of the National antidiabetic activity [1, 4], with their beneficial effects Institute of Chemical-Pharmaceutical R&D (ICCF being attributed to the copresence of sitosterol, inulin Bucharest), Romania. The burdock leaves were shade and lignans[2]. Furthermore, the combination arctigenin dried and ground to a medium-size plant powder; voucher - polyphenols have shown the capacity to block tumor cell specimens (ALam7.1, Alam7.2) are deposited in the ICCF growth and metastasis [13]. Additionally, arctiin, caffeic Plant Material Storing Room. and chlorogenic acid mixtures have proven significant anti-mutagenic effects and a positive correlation with polyphenolic content [14]. Concerning the biochemical 2.1.2 Vegetal extracts preparation pathway involved, studies on arctigenin tested on MH60 cells indicated the stimulation of apoptosis (IC50 = 1.0 μM) Fifty (50) g of leaf powder (Arctii folium) were heat-assisted as being the most probable mechanism [15], while the extracted (twice) with 500 mL of 70% (v/v) ethanol. The capacity of polyphenols to protect the cells against radical total extract obtained was filtered through a medium( ) oxygen species (ROS) injuries and to stimulate immune paper filter, resulting in 650 mL ethanol extract (E7). Two processes [16] are believed to effectively contribute to the hundred and fifty (250) mL of (E7) ethanol extract was antitumor effect of burdock products. concentrated at low pressure at sicc product then passed The leaves of Arctium lappa L. have been recognized into 20% (v/v) propylene glycol (20% PG) solution so as havingsome antimicrobial properties against some as to ensure a final concentration of exactly 5 mg total bacterial strains found in endodontic infections such phenols (expressed as gallic acid [GAE] equivalents) per as Bacillus subtilis, Lactobacillus acidophilus and 1 mL extract, further called Arctii folium whole ethanol Pseudomonas aeruginosa, as well as on Candida albicans, extract (codified W); the whole ethanol extract (W) was Escherichia coli, Staphylococcus aureus and Micrococcus divided into 2 mL Eppendorf tubes and stored at -8 °C until luteus [12]. Furthermore, the efficacy of burdock leaf microbiological studies. Another two hundred and fifty derived products against influenza A virus (A/NWS/33, (250) mL of (E7) ethanol extract was also concentrated at H1N1) (IFV) [17], herpesvirus (HSV-1, HSV-2), adenovirus sicc product then dissolved into 100 mL of distilled water. (ADV-3, ADV-11), and human immunodeficiency virus The resulting aqueous solution was (manually) extracted (HIV-1) [18] has also been proven. first with (3 x 100 mL) chloroform and then with (3 x 100 Thus, in the current upward trend of using plant mL) ethyl acetate solvent, 24 hours per each stage. Three derived products as natural medicines, while at the same selective extracts were obtained: aqueous (AQ), ethyl time increasing bacterial resistance to antibiotics of all acetate (EA) and chloroform (CHL) fractions, respectively. classes, based on these data, but also on our previous The three Arctii folium selective fractions were analyzed as results [19, 20] demonstrating the antimicrobial effects of concerning total phenols content (GAE per 1 mL sample) some whole and selective extracts from medicinal plants then concentrated at sicc product and (separately) passed 94 Lucia Pirvu et al. into 20% PG as follows: aqueous and ethyl acetate ATCC 29245, Escherichia coli ATCC 35218, E. coli ATCC fractions were prepared so as to achieve identical 5 mg 11229, E. coli ATCC 8739, and Bacillus cereus ATCC 11778). GAE / 1 mL samples, while the chloroform fraction (CHL) Nutrient broths (Oxoid CM0001) and Muller-Hinton agar was passed into 20% PG by means of the whole extract (Oxoid CM0337) culture media were used (prepared from algorithm so as to assure the same amount of non-polar a dehydrated base according with the manufacturer’s compounds. Similarly, the three selective extracts, AQ, EA recommendations). and CHL (test extracts) were divided into monodoses and stored at -8 °C until microbiological studies. 2.2.3.2 Test antimicrobials Six antibiotics (microtablets purchased from Biolab Zrt. 2.1.3 Chemicals, reagents and references Romania) were used: Ampicillin (AM, 10 μg), Gentamicin (CN, 10 μg), Tetracycline (TE, 30 μg), Sulfamethoxazole/ Reagents (Folin-Ciocalteau and Natural Product) and Trimethoprim (SXT, 25 μg), Ciprofloxacin (CIP, 5 μg), and solvents (methanol, ethanol, ethyl acetate, formic acid, Chloramphenicol (C, 30 μg). glacial acetic acid and chloroform) were purchased from Sigma-Aldrich Co, Fluka and Biochemika, as well as quercetin (95%), rutin (min. 95%), quercitrin (>90%), 2.2.3.3 Inoculum preparation isoquercitrin (>90%), hyperoside (>97%), apigenin The microorganisms were stored in freezing conditions (>97%), vitexin-2’’O-rhamnoside (>98%), apigenin 7-O- and activated by cultivation in Nutrient broth at 37 °C apiosylglucoside/apiin (>99%), chlorogenic acid (>95%), for 24 hours. Bacterial inoculums were prepared from an rosmarinic acid (97%), caffeic acid (>98%) and cynarin overnight broth culture in demineralised water in order to (analytical standard) polyphenols compounds – the obtain a bacterial turbidity equivalent to 0.5 McFarland reference substances (ref.). standards [24].

2.2 Experimental Design 2.2.3.4 Minimum inhibitory concentration (MIC) assay The experiments were accomplished using the reference 2.2.1 Qualitative (HP)TLC analysis method for testing the in vitro activity of the antimicrobial agents, ISO 20776-1/2006 [25] and CLSI M07-A9 document Studies were performed using TLCand a Linomat 5, CAMAG [26]. MIC appraisals were carried out using an accurate apparatus, (Muttenz – Switzerland) according to a general working scheme (see Table 1S) as follows: four identical method for the assessment of polyphenols (Wagner and Arctii folium dilution series were prepared, corresponding Bladt [21] and Reich and Schibli [22]), as described in to four test extracts, i.e. the whole ethanol extract/W previous studies [19, 20]. and the three selective fractions/AQ,EA, CHL, each one precisely quantified as concerning total phenols content (μg GAE / 1 mL sample); the dilution series were fulfilled 2.2.2 Estimation of Total Phenolics Content by incorporation of each vegetal extract into Muller- Hinton Agar (MHA) medium so that the concentration Studies were performed according to the Folin-Ciocalteu level of the four test extracts (dilution series) ranged in method [23], also detailed in previous studies [19, 20]. the interval 4 – 1024 μg GAE / mL in case of the polar extracts (W, AQ and EA) and 0.1 – 25.6 μg GAE / mL in case of nonpolar extract (CHL); MHA medium + PG 20% 2.2.3 Antimicrobial Activity Assay solution (4.1 ml + 0.9 ml) was used as the negative control. After the solidification of the MHA mixed with the tested 2.2.3.1 Test organisms vegetal extract, the bacterial inoculum (104 cfu/spot) Nine reference bacterial strains (ATCC strains purchased was applied to the surface of the medium and the plates from Thermo ScientificRomania) were used, including were incubated for 24 hours at 37 °C. The results were four Gram-positive bacteria (Staphylococcus aureus ATCC interpreted according to literature data [27] by evaluating 6538, Staphylococcus aureus ATCC 25923, Enterococcus the bacterial growth at respective vegetal extract dilution faecalis ATCC 29212, and Staphylococcus epidermidis ATCC series and concentration of phenols. 12228) and five Gram-negative bacteria Proteus( mirabilis Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 95

2.2.3.5 Synergism assay Rf≈0.68/0.64), isoquercitrin (s4/Rf≈0.69/0.71), quercitrin The assessment of potential interactions between the four (s5/Rf≈0.77), cynarin (s5’/Rf≈0.77) and another major Arctii folium extracts and the six antibiotics (microtablets caffeoylquinic acid isomer(s6/Rf≈0.90/0.92 – the major of accurate concentration) has as scientific basis and compound in ethyl acetate fraction/EA) attributed to 1,5- further design study model their combination at the MIC/2 di-O-caffeoyl-4-O-maloylquinic acid [4]. concentration level [28]. Thus, the culture medium (MHA) Furthermore, some differences were revealed between was prepared by incorporating Arctii folium extracts so the extracts obtained from leaves collected at different that the polyphenols have a concentration corresponding to MIC/2. The dried surface of the culture medium was thus inoculated with a bacterial suspension at 1-2x108 UFC / mL and subsequently the antimicrobial micro-tablets were distributed to achieve MIC/2 concentration level as well. The plates were incubated for 24 hours at 37 °C, and then the antimicrobial effect was evaluated by measuring the inhibition area around the microtablets. For each antimicrobial substance (positive control/C), four combinations were designated: C+Whole extract (C+W), C+Aqueous extract (C+AQ), C+Ethyl acetate fraction (C+EA) and C+ Chloroform fraction (C+CHL).

2.2.3.6 Statistical analysis Results are expressed as the mean ± SD of three experiments; significant stimulatory effect between the antimicrobial substance and test extract was considered when the combined inhibition zone’s diameter enlarged by at least 5 mm.

3 Results and Discussion

3.1 Analytical aspects of Arctii folium extracts

Chemical qualitative aspects and the polyphenols content, of Arctium lappa whole and selective leaf extracts have been established by using an (HP)TLC method. Studies (Figure Figure 1: TLC aspects of Arctium lappa folium extracts comparatively 1 – chromatograms A1, A2 and A3) indicated the three to the reference substances (ref.). A1: Track T1, quercetin-3-O- polar extracts (the whole ethanol extract/W, ethyl acetate rutinoside/ rutin, chlorogenic acid, apigenin-7-O-apiosyl-glucoside/ fraction/EA and aqueous fraction/AQ) as containing two apiin, quercetin-3-O-galactoside/ hyperoside, rosmarinic acid, main polyphenol subclasses, the caffeoylquinic acid and apigenin (ref); Tracks T2, burdock leaves whole (70%, v/v) isomers (blue fluorescent/fl. spots - s2, s3’, s5’ and s6) and ethanol extract/W – duplicate sample; Track T3, rutin, vitexin-2’’- O-rhamnoside, luteolin-7-O-glucoside/ cynaroside, quercetin-3- quercetin derivates (orange fl. spots - s1, s3, s4 and s5) and O-rhamnoside/ quercitrin, and quercetin (ref.); A2: Tracks T2aq, the nonpolar, chloroform fraction/CHL, which did not burdock leaves aqueous fraction/AQ – duplicate sample; Track reveal any polyphenol compounds. Based on the literature T2ea, burdock leaves ethyl acetate fraction/EA – duplicate sample; data [5], but also on the current reference substances/ref. Track T2chl, burdock leaves chloroform fraction/CHL – single used (see T4, T5, T6, T7 and T8 tracks), the main spots sample. A3: Track T4, rutin, chlorogenic acid, caffeic acid (ref); Track T5, quercetin-3-O-glucoside/ isoquercitrin (ref); Track T6, quercetin- were attributed to rutin (s1/Rf≈0.43), chlorogenic acid 3-O-galactoside/ hyperoside (ref); Track T2ea, burdock leaves ethyl (s2/Rf≈0.52/0.55 – the major compound in the whole acetate fraction/EA; Track T2aq, burdock leaves aqueous fraction/ ethanol extract/W and subsequent aqueous fraction/AQ), AQ; Track T2, burdock leaves whole ethanol extract/W; Track T7, neochlorogenic acid (s3’/Rf≈0.61/0.64) hyperoside (s3/ cynarin (ref). Track T8; caffeic acid (ref). 96 Lucia Pirvu et al.

Table 1: MIC values of the four Arctii folium extracts tested on nine ATCC bacterial strains.

No. Bacterial strain MIC (μg GAE / mL) Control sample crt. (MHA + PG) Whole Aqueous Ethyl acetate Cloroform extract (W) fraction (AQ) fraction (EA) fraction (CHL) 1. Staphycoccus aureus ATCC 6538 512 256 1024 512* positive 2. Staphycoccus aureus ATCC 25923 256 1024 1024 1024* positive 3. Staphylococcus epidermidis ATCC 12228 128 32 512 1024* positive 4. Proteus mirabilis ATCC 29245 256 512 512 1024* positive 5. Escherichia coli ATCC 35218 > 1024 1024 1024 > 1024* positive 6. Escherichia coli ATCC 11229 > 1024 1024 1024 > 1024* positive 7. Escherichia coli ATCC 8739 > 1024 1024 1024 > 1024* positive 8. Bacillus cereus ATCC 11778 512 1024 512 1024* positive 9. Enterococcus faecalis ATCC 29212 1024 1024 1024 > 1024* positive

*Note: Based on the analytical quantitative studies indicating 0.125 mg GAE / mL chloroform fraction/sample, 512* and (>)1024* values represents the total phenols content (expressed as μg GAE / mL sample) of the CHL fraction calculated for identical dilution series with the whole extract (W). vegetation times; Arctii folium derived products from fraction/AQ and the whole extract/W), with MIC values September (chromatogram A3) did not reveal the presence measuring 32 and 128 μg GAE / mL sample. of quercetin-3-O-rutinoside/rutin and corresponding On P. mirabilis ATCC 29245 and S. aureus ATCC 25923 as monoglycoside, quercetin-3-O-rhamnoside/quercitrin, well as on S. aureus ATCC 6538, the whole extract/W and previously seen [19] in Arctii folium extracts during the the aqueous fraction/AQ indicated identical MIC values of June period (chromatograms A1 and A2). 256 μg GAE / mL sample. Regarding chemical quantitative aspects, the whole The results also reveal the lack of activity of all tested ethanol extract/W, and aqueous/AQ and ethyl acetate/EA extracts against B. cereus ATCC 11778, E. faecalis ATCC selective extracts were made to provide the exact content 29212 and the three E. coli ATCC strains, as well as the of 5 mg total phenols (GAE) / mL sample. inefficiency of the nonpolar chloroform fraction (CHL) on Designed so as to ensure identical levels of the non- all microbial strains studied. polar compounds as the whole extract/W, the selective Furthermore, the nine bacterial strains properly chloroform extract/CHL also indicated small amounts of developed on MHA and MHA plus (20%, v/v) propylene phenolic compounds (0.125 mg GAE / mL sample), even if glycol solution (MHA + PG) medium, thus proving the lack not confirmed by TLC studies. of influence on the bacterial growth positive( control).

3.2 Evaluation of the minimum inhibitory 3.3 Effects of Arctii folium extracts upon (six) concentration (MIC) antibiotics

Defined as the lowest concentration of an antimicrobial Studies have been done on six antibiotics (Ampicillin/AM, agent that, under definedin vitro conditions, prevents the Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole- appearance of visible growth of the microorganism after Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/ its incubation overnight (CLSI, ISO), minimum inhibitory CN), each one belonging to a separate class of antibiotic, concentration (MIC) appraisal was carried out using with the antimicrobial used being classed by the official an accurate working scheme (Table 1S) and precisely reports of the European Union as effective [29], and in the quantified test vegetal samples as concerning the total same time being extensively used for treating infections phenols content (μg GAE per 1 mL sample). worldwide. The results (Table 1) suggest reserved antimicrobial As a working principle, all combinations (antibacterial potency of Arctii folium extracts, the best results being substance - vegetal extract) were studied on all bacterial shown on S. epidermidis ATCC 12228 (precisely the aqueous strains (Table 2), excepting those combinations and strains Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 97

Table 2: The effects of Arctii folium extracts on the antimicrobial activity of the six antibiotics tested on the nine microbial strains, diameter of the inhibition zone (mm) ± SD respectively.

Tested sample Diameter of the inhibition zone (mm) ± SD

S. S. aureus S. P. mirabilis E. coli E. coli E.coli 8739 B. E. aureus 25923 epidermidis 29245 35218 11229 cereus faecalis 6538 12228 11778 29212 Ampicillin (AM) combinations AM (C) 32±0.86 30±1.15 19±0.86 17±0.57 6±0.00 7±0.00 6±0.00 6±0.00 19±0.86 AM+W 40±1.80 42±1.73 19±1.00 15±0.57 ND ND ND 11±1.52 25±1.32 AM+AQ 36±1.73 30±1.52 15±0.57 18±0.86 6±0.00 15±0.57 6±0.00 14±0.57 27±1.73 AM+EA 36±2.17 40±1.32 19±0.86 16±1.00 6±0.00 18±1.00 6±0.00 8±0.00 26±0.86 AM+CHL 38±1.00 36±2.02 14±1.32 17±1.73 ND ND ND 15±0.57 ND Tetracycline (TE) combinations TE (C) 27±1.73 27±1.15 10±1.00 6±0.00 20±1.73 19±0.86 22±1.73 28±1.00 9±1.00 TE+W 36±1.73 36±2.17 10±1.00 8±0.00 ND ND ND 37±1.00 17±0.57 TE+AQ 34±1.00 30±1.15 8±1.00 7±0.00 27±1.73 25±0.57 28±1.32 40±1.80 15±0.57 TE+EA 37±0.86 34±1.32 13±0.57 7±0.00 36±1.73 34±1.73 30±1.32 32±0.86 19±0.86 TE+CHL 28±1.32 26±1.32 11±0.57 6±0.00 ND ND ND 30±1.52 ND Ciprofloxacin (CIP) combinations CIP (C) 34±1.32 32±1.00 27±1.73 39±1.73 37±1.73 28±1.73 40±1.32 28±1.52 22±1.73 CIP+W 40±1.00 42±1.15 27±0.57 47±1.52 ND ND ND 35±1.73 26±1.73 CIP+AQ 34±1.73 26±0.57 25±0.57 37±1.73 30±1.15 27±1.73 33±1.73 26±1.32 23±1.00 CIP+EA 36±1.32 40±1.80 28±1.73 43±0.57 37±0.50 32±0.86 40±1.80 29±0.57 21±1.00 CIP+CHL 39±1.73 36±1.73 26±1.00 37±1.00 ND ND ND 42±1.15 ND Sulfamethoxazole-Trimethoprim (SXT) combinations SXT (C) 28±1.00 28±1.73 25±1.32 25±1.00 20±1.73 21±0.57 24±0.50 6±0.00 25±1.32 SXT+W 35±1.73 32±0.86 25±1.00 35±1.73 ND ND ND 6±0.00 29±1.32 SXT+AQ 28±1.73 25±0.57 25±1.32 27±1.73 18±1.00 21±0.86 22±1.00 6±0.00 24±0.50 SXT+EA 37±0.86 32±0.86 18±0.86 27±1.15 18±0.57 20±0.57 24±1.73 6±0.00 18±1.00 SXT+CHL 29±0.86 30±0.86 24±0.86 23±2.08 ND ND ND 6±0.00 ND Chloramphenicol (C) combinations C (C) 26±1.32 25±0.57 25±0.57 18±1.00 8±0.00 19±0.86 10±0.86 23±2.08 19±2.29 C+W 35±1.73 36±1.73 25±1.32 21±1.73 ND ND ND 29±0.86 27±1.73 C+AQ 32±0.86 27±1.73 24±0.50 18±0.86 8±0.00 28±1.73 6±0.00 28±1.00 28±1.73 C+EA 34±1.73 35±1.73 25±1.00 21±1.32 11±0.57 26±1.00 6±0.00 25±1.32 28±1.73 C+CHL 32±0.86 29±1.73 24±1.00 20±1.00 ND ND ND 32±0.86 ND Gentamicin (CN) combinations CN (C) 26±1.32 28±1.73 19±0.86 22±1.52 20±1.00 18±1.00 22±1.73 25±0.57 14±1.32 CN+W 33±1.73 36±1.73 20±1.73 28±1.73 ND ND ND 31±1.52 20±1.00 CN+AQ 26±1.32 22±1.52 18±1.00 21±1.32 18±1.00 21±1.00 22±1.00 25±1.00 14±1.00 CN+EA 23±1.32 34±1.73 17±0.57 23±1.00 25±1.32 21±1.32 22±1.73 25±0.57 15±0.57 CN+CHL 28±1.00 28±1.73 18±1.00 22±1.73 ND ND ND 28±1.00 ND

Where: ND = Not determined; W = Whole extract, AQ = Aqueous fraction, EA = Ethyl acetate fraction, CHL = Chloroform fraction, the four vegetal extracts used in combination with the six antibiotics respectively. 98 Lucia Pirvu et al. for which MIC values could not be precisely determined; these cases were pointed out as ND (not determined). For each antimicrobial substance (positive control/C), four combinations with the vegetal extracts (C+W, C+AQ, C+EA and C+CHL) were tested, and a signifi cant stimulatory eff ect between the antimicrobial substance and the test extract was considered when the combined inhibition zone’s diameter enlarged by at least 5 mm [30]. Figures 2 – 7 show the bacterial response after the treatment with respective vegetal-chemical antimicrobial combination, antibiotics’ effi cacy on the nine microbial strains respectively. Therefore, compared to the antibiotic alone (positive Figure 2: Bacterial strains response to Ampicillin/AM combinations with the four Arctii folium extracts (AM+W, AM+AQ, AM+EA, and control/C), Ampicillin/AM (Figure 2) and Arctii folium AM+CHL) comparatively to the antibiotic alone, AM (C); diameter of extracts showed the best results against E. coli ATCC 11229 the inhibition zone, (mm) ± SD. and B. cereus ATCC 11778 strains. Ampicillin’s combinations with Arctii folium extracts were also eff ective on the two S. aureus strains (ATCC 6538 and ATCC 25923) and E. faecalis ATCC 29212, while on S. epidermidis ATCC 12228, P. mirabilis ATCC 29245 and the other two E. coli strains (ATCC 35218 and ATCC 8739), only inhibitory eff ects or the lack of activity were observed. Tetracycline/TE (Figure 3) and Arctii folium polar extracts (W, AQ and EA) showed good cooperation in making fi nal antimicrobial activity, on all microbial strains. The most important stimulatory eff ects were noticed on E. faecalis and the three E. coli strains (EA and AQ), but also on B. cereus, the two S. aureus strains and P. mirabilis. Ciprofl oxacin/CIP Figure ( 4) combined with Arctii Figure 3: Bacterial strains response to Tetracycline/TE combinations folium extracts demonstrated stimulatory, but inhibitory, with the four Arctii folium extracts (TE+W, TE+AQ, TE+EA, and eff ects too. The most important stimulatory eff ects were TE+CHL) comparatively to the antibiotic alone, TE (C); diameter of provided by the whole extract (W) in case of B. cereus ATCC the inhibition zone, (mm) ± SD. 11778 and S. aureus ATCC 25923, while the most important inhibitory eff ects have been noticed in case of the aqueous fraction/AQ (appraised up to -19% inhibitory eff ect). Sulfamethoxazole-Trimethoprim/SXT (Figure 5) combinations (mainly with the whole extract/W) resulted in stimulatory eff ects upon P. mirabilis ATCC 29245, both S. aureus strains and E. faecalis ATCC 29212, the other microbial strains being practically unreactive to this combination. Similar to CIP combinations, the aqueous fraction/AQ as well as ethyl acetate fraction/EA along with SXT induced numerous inhibitory eff ects, mainly on S. epidermidis ATCC 12228 (EA lead up to -28% inhibitory eff ect). Chloramphenicol/C (Figure 6) gained substantial effi cacy after combining with Arctii folium polar and Figure 4: Bacterial strains response to Cyproﬂ oxacin/CIP combinations with the four Arctii folium extracts (CIP+W, CIP+AQ, non-polar extracts on all tested bacteria, excepting CIP+EA, and CIP+CHL) comparatively to the antibiotic alone, CIP (C); S. epidermitis ATCC 12228 and E. coli ATCC 8739; the most diameter of the inhibition zone, (mm) ± SD. important synergistic activities were obtained against E. faecalis ATCC 29212 (induced by polar extracts), both Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 99

S. aureus strains and B. cereus ATCC 11778 (induced by polar and non-polar extracts), as well as upon E. coli ATCC 35218 and E. coli ATCC 11229 (induced by aqueous/AQ and/or ethyl acetate/EA fractions). E. coli ATCC 8739 strain indicated prominent inhibitory eff ects by combining with AQ and EA fractions (appraised as -40% for both cases). Gentamicin/CN (Figure 7) along with Arctii folium whole extract/W lead to stimulatory eff ects, the other vegetal extracts resulting in less augmented stimulatory but inhibitory eff ects (mainly induced by aqueous/AQ and ethyl acetate/EA fractions) too. The most susceptible bacterial strains to CN combinations with the whole Figure 5: Bacterial strains response to Sulfamethoxazole- extract/W were E. faecalis 29212, followed by the two Trimethoprim/SXT combinations with the four Arctii folium extracts S. aureus strains, P. mirabilis ATCC 29245 and B. cereus (SXT+W, SXT+AQ, SXT+EA, and SXT+CHL) comparatively to the ATCC 11778; an important inhibitory eff ect of the aqueous antibiotic alone, SXT (C); diameter of the inhibition zone, (mm) ± SD. fraction/AQ was recorded against S. aureus 25923 (-21% inhibitions). In terms of stimulation and/or inhibition percentage improvement compared to the antibiotic alone, the relative ratio (%) computed respectively (see Table 2S), certain increases in susceptibility of the S. aureus ATCC 6538 strain (Figure 1S) by combining TE, C and SXT with Arctii folium extracts was clearly seen, mainly with the whole extract/W and ethyl acetate/EA fraction. Proving these, Tetracycline/ TE combined with W and EA showed the highest capacity to increase the antimicrobial effi cacy leading up to 35% and, respectively, 37% stimulatory eff ects compared to the antibiotic alone (this meaning up to 12% and, respectively, 16% synergistic activity), Chloramphenicol/C led to 35% Figure 6: Bacterial strains response to Chloramphenicol/C and 31% stimulatory eff ects compared to the antibiotic combinations with the four Arctii folium extracts (C+W, C+AQ, C+EA, alone therefore appraised as 12% and 10% synergistic and C+CHL) comparatively to the antibiotic alone, C (C); diameter of activity; Sulfamethoxazole-Trimethoprim/SXT led to 25% the inhibition zone, (mm) ± SD. and 32% stimulatory eff ects compared to the antibiotic alone further calculated as 6% and, respectively, 12% synergistic activity. The S. aureus ATCC 25923 strain (Figure 2S) also indicated increased susceptibility after combining tested antibiotics with the whole extract/W and ethyl acetate/EA fraction, Chloramphenicol/C, Ampicillin/ AM, Ciprofl oxacin/CIP and Tetracycline/TE combinations indicating the best cooperation in making the fi nal antimicrobial activity (it was measured up to 44% antimicrobial effi cacy boost compared to the antibiotic alone meaning up to 20% synergistic activity). Regarding the S. epidermitis ATCC 12228 strain, most of the studied antimicrobial combinations led to Figure 7: Bacterial strains response to Gentamicin/CN combinations inhibitory eff ects or no eff ects; the only exception was with the four Arctii folium extracts (CN+W, CN+AQ, CN+EA, and that of Tetracycline/TE which showed stimulatory eff ects CN+CHL) comparatively to the antibiotic alone, CN (C); diameter of the inhibition zone, (mm) ± SD. when combined with the EA fraction. It must be noticed that Arctii folium polar extracts (AQ and W) indicated a 100 Lucia Pirvu et al. certain antimicrobial activity on S. epidermidis 12228 (MIC Table 3. General view of the effects of Arctii folium extracts on the values measuring 32 and 128 μg GAE / mL sample), so six antibiotics tested. that the lack of synergistic activity of Arctii folium extracts along with antibiotics tested convey the normal behavior Type of effect Antibiotic tested / number of cases of a synergistic product. The P. mirabilis ATCC 29245 strain (Figure 3S) proved to AM TE CIP SXT C CN be sensitive especially to the whole extract/W combination Lack of interaction (meaning 15 10 11 16 11 15 identical Ø and ND situations) with SXT (the stimulatory effect was appraised up to 40% compared to the antibiotic alone meaning 17% synergistic Inhibitory effect 4 1 10 10 4 7 activity), but also with CIP (up to 20% stimulatory effect Stimulatory effect 17 25 15 10 21 14 compared to the antibiotic meaning 7% synergistic activity) Stimulatory effect over 5 mm 12 16 6 3 13 6 and CN (up to 28% stimulatory effect compared to the Ø (synergistic effects) antibiotic meaning 4% synergistic activity) antibiotics. Studies on the three E. coli strains have indicated several prominent synergistic effects, precisely when combining aqueous/AQ and ethyl acetate/EA fractions with TE, AM or C antibiotics. Thus, E. coli ATCC 35218 (Figure 4S) has been very sensitive to TE combinations with AQ and EA leading up to 35% and, respectively, 80% antimicrobial efficacy increases compared to the antibiotic alone further estimated as 8% and, respectively, 44% synergistic activities. E. coli ATCC 11229 (Figure 5S) also reacted to AM combinations with AQ and EA fractions leading up to 110% and 160% antimicrobial efficacy increases compared to the antibiotic alone estimated as 25% and 50% synergistic activities. E. coli ATCC 11229 has also been sensitive to TE and C combinations with the two polar extracts, AQ and EA, leading to 4 and 42% and, respectively, 17 and 8% synergistic activities. E. coli ATCC 8739 (Figure 6S) reacted to TE combinations with AQ and EA fractions achieving 4% and 11% synergistic activities. B. cereus ATCC 11778 strain (Figure 7S) has revealed a high susceptibility to AM, TE and CIP combinations with AQ, W and CHL extracts, and less effective with the EA fraction; thus, AM and Arctii folium extracts lead to an antimicrobial efficacy increase of 133% when combined with AQ (meaning 27% synergistic activity) and of 150% when in combination with CHL (36% synergistic activity); TE and Arctii folium extracts lead to an increase in antimicrobial efficacy of 25% when combined with W (12% synergistic activity) and of 40% when combined with AQ (21% synergistic activity); CIP and Arctii folium extracts led to an antimicrobial efficacy increase of 25% when combined with the whole extract/W (9% synergistic activity) and of 50% in combination with the chloroform fraction/CHL (27% synergistic activity). E. faecalis ATCC 29212 (Figure 8S) showed increased Figure 8. The situation upon specific antibiotic a) and individual sensitivity after combining TE, C and AM with Arctii folium extract b). all polar extracts (W, AQ and EA): TE combinations led Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 101 to 89%, 67%, and 111% antimicrobial efficacy increases (CHL), this could be active on the basis of its non-polar compared to the antibiotic alone meaning 21%, 7%, and status thus allowing a better internalization of the active 36% synergistic activities; C combinations led up to 42%, compounds into bacteria. 47%, and 47% antimicrobial efficacy boosts and 12%, 17%, Finally, the results may be useful for physicians and and 17% synergistic activities, while AM combinations lead pharmacists who are currently using phytomedicines up to 32%, 42%, and 37% antimicrobial efficacy boosts and plant based medicines, in order to avoid potential and 4%, 12%, and 8% synergistic activities respectively. interactions between Arctii folium derived products and Table 3 and Figure 8 (a and b) cumulate the results antibiotics. obtained by testing the 24 pairs of chemical–vegetal antimicrobial combinations on the nine microbes. Acknowledgements: The authors from National Institute Thus, with 22 synergistic interactions (meaning of Chemical Pharmaceutical Research and Development significant biological activity certified by a combined (ICCF) - Bucharest Romania, gratefully acknowledge the inhibition zone exceeding 5 mm diameter) and only one financial support from project POC ID P_40_406, SMIS situation of inhibitory effect,Arctii folium whole (70%, 105542 sustaining the design and development of plant v/v) ethanol extract/W with exactly 5 mg gallic [GAE] acid derived products, their chemical characterization and equivalents per 1 mL sample appear as the most realistic data processing. synergistic product along with the six (susceptible) antimicrobials tested, mainly with the Tetracycline/TE Conflict of interest:The authors declare no competing antibiotic. financial interest.

4 Conclusions References [1] Jingvi L., Yi-Zhong C., Ricky N.S.W., Calvin, K.F.L., Sydney Therefore, based on the present study, three conclusions C.W.T., Stephen C.W.S., Yao T., Yanbo Z., Comparative Analysis and potential uses have been drawn: first of all, the of Caffeoylquinic Acids and Lignans in Roots and Seeds unpredictable effect (stimulatory or inhibitory) ofArctii among Various Burdock (Arctium lappa) Genotypes with High folium derived products (mainly the aqueous extract/ Antioxidant Activity, J. Agric. Food Chem., 2012, 60, 4067– 4075. AQ) in combination with current antibiotics has been [2] Chan Y.S., Cheng L.N., Wu J.H., Chan E., Kwan Y.W., Lee demonstrated; secondly, the results suggest potential S.M.Y., Leung G.P.H., Yu P.H.F., Chan S.W., A review of uses of Arctii folium whole (70%, v/v) ethanol extract the pharmacological effects of Arctium lappa (burdock), in restoring the activity of the antibiotics affected by Inflammopharmacology, 2011, 19, 245-254. microbial resistance; lastly (and also backed up by our [3] Yoshihiko M., Jun K., Ryoya N., Antioxidative caffeoylquinic acid previous studies on Epilobi hirsuti herba extracts [20]), derivatives in the roots of burdock (Arctium lappa L.), J. Agric. Food Chem., 1995, 43, 2592–2595. further studies on separate compounds and flavonols [4] Tousch D., Bidel L.P.R., Cazals G., Ferrare K., Leroy J., Faucanie (eg., myricetin and quercetin derivates [31]) combinations M., Chevassus H., Tournier M., Lajoix A.D., Azay-Milhau J., with phenylcarboxilic acids (e.g., gallic and caffeic acid Chemical Analysis and Antihyperglycemic Activity of an Original derivates) are proposed as a starting point for developing Extract from Burdock Root (Arctium lappa), J. Agric. Food new, natural synergistic agents useful in the antibiotic Chem., 2014, 62, 7738–7745. [5] Ferracane R., Graziani G., Gallo M., Fogliano V., Ritieni A., industry. Found to be present in all of the polar extracts Metabolic profile of the bioactive compounds of burdock (W, AQ and EA), the two main quercetin monoglycosides, (Arctium lappa) seeds, roots and leaves, Journal of hyperoside (s3) and isoquercitrin (s4) respectively, are the Pharmaceutical and Biomedical Analysis, 2010, 51, 399-404. most probable active (synergistic) compounds in Arctii [6] Tita I., Mogosanu G.D., Tita M.G., Ethnobotanical inventory of folium derived products. While the quantity and rate of medicinal plants from the south-west of Romania, Farmacia, these two quercetin monoglycosides, as well as the co- 2009; 57, 141-156. [7] Shimizu J., Yamada N., Nakamura K., Takita T., Innami S., presence of different quantity and quality of phenolic acids Effects of different types of dietary fiber preparations isolated could explain the similarities, but also the differences from bamboo shoots, edible burdock, apple and corn on between the three polar vegetal extracts, the microbial profiles of rats, Journal of Nutritional Science and Vitaminology, susceptibility to antibiotics and vegetal compounds could 1996, 42, 527-39. explain the final reaction of bacteria to each combination [8] Grases F., Melero G., Costa-Bauza A., Prieto R., March J.G., Urolithiasis and phytotherapy, International Urology and studied. Regarding the non-polar, chloroform fraction Nephrology, 1994, 26, 507-11. 102 Lucia Pirvu et al.

[9] Knott A., Reuschlein K., Mielke H., Wensorra U., Mummert C., [21] Wagner H., Bladt S., Plant Drug Analysis, Second Ed., Springer, Koop U., Kausch M., Kolbe L., Peters N., Stäb F., Wenck H., Berlin, 1996. Gallinat S., Natural Arctium lappa fruit extract improves the [22] Reikh E., Schibli A., HPTLC for the Analysis of Medicinal Plants, clinical signs of aging skin, J Cosmet Dermatol., 2008, 7, 281- Thieme, N.Y. Stuttgart, 2008. 289. [23] Romanian Pharmacopoeia, Xth ed., Ed. Medicala, Bucharest, [10] Maghsoumi-Norouzabad L., Alipoor B., Abed R., Eftekhar S.B., 1993. Mesgari-Abbasi M., Asghari J.M., Effects of Arctium lappa L. [24] Lakis Z., Mihele D., Nicorescu I., Vulturescu V., Udeanu (Burdock) root tea on inflammatory status and oxidative stress D.I., The antimicrobial activity of Thymus vulgaris and in patients with knee osteoarthritis, Int J Rheum Dis., 2016, 19, Origanum syriacum essential oils in Staphylococcus aureus, 255-261. Streptococcus pneumoniae and Candida albicans, Farmacia, [11] Pereira J.V., Bergamo D.C., Pereira J.O., Antimicrobial activity of 2012, 6, 857-865. Arctium lappa constituents against microorganisms commonly [25] CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for found in endodontic infections, Braz Dent J., 2005, 16, Bacteria that Grow Aerobically; Approved Standard - 9th Edition. 192–196. CLSI document M07-A9. Wayne, PA: Clinical and Laboratory [12] Gentil M., Pereira J.V., Silva Sousa Y.T.C., Pietro R., Sousa Neto Standards Institute, 2012. M.D., Vansan L.P., Suzelei de Castro F., In vitro evaluation of the [26] ISO 20776-1/2006. Clinical laboratory testing and in vitro antibacterial activity of Arctium lappa as a phytotherapeutic diagnostic test systems – Susceptibility testing of infectious agent used in intracanal dressings, Phytotherapy Research, agents and evaluation of performance of antimicrobial 2006, 20, 184-186. susceptibility test devices – Part 1: Reference method for [13] Awale S., Lu J., Kalauni S.K., Kurashima Y., Tezuka Y., Kadota testing the in vitro activity of antimicrobial agents against S., Esumi, H. Identification of arctigenin as an antitumor agent rapidly growing aerobic bacteria involved in infectious having the ability to eliminate the tolerance of cancer cells to diseases. International Organization for Standardization, nutrient starvation, Cancer Res., 2006, 66, 1751-1757. Geneva, Switzerland, 2006. [14] Liu L., Tang L., Studies on antimutagenicity of Burdock, Acta [27] Andrews J.M., Determination of minimum inhibitory Academiae Medicine Nanjing, 1997, 4, 343-345. concentrations, Journal of Antimicrobial Chemotherapy, 2001, [15] Matsumoto T., Hosono-Nishiyama K., Yamada H., 48, 5-16. Antiproliferative and Apoptotic Effects of Butyrolactone Lignans [28] Noumedem J.A.K., Mihasan M., Kuiate J.R., Stefan M., Cojocaru from Arctium lappa on Leukemic, Planta Med., 2006, 72, 276- D., Dzoyem J.P., Kuete V., In vitro antibacterial and antibiotic- 278. potentiation activities of four edible plants against multidrug- [16] Miyamoto K., Nomura M., Sasakura M., Matsui E., Koshiura resistant gram-negative species, BMC Complementary and R., Murayama T., Furukawa T., Hatano T., Yoshida T., Okuda Alternative Medicine, 2013, 13, 190-199. T., Antitumor-Activity of Oenothein-B, a Unique Macrocyclic [29] European Food Safety Authority (EFSA) & European Centre for Ellagitannin, Jpn. J. Cancer Res.,1993, 84, 99-103. Disease Prevention and Control (ECDC), EU Summary Report [17] Hayashi K., Narutaki K., Nagaoka Y., Hayashi T., Uesato on antimicrobial resistance in zoonotic and indicator bacteria S., Therapeutic effect of arctiin and arctigenin in from humans, animals and food in 2013. EFSA Journal 2015, 13, immunocompetent and immunocompromised mice infected 4036. with influenza A virus, Biol Pharm Bull., 2010, 33, 1199-1205. [30] Ahmad I., Aqil F., In vitro efficacy of bioactive extracts of 15 [18] Eich E., Pertz H., Kaloga M., Schulz J., Fesen M.R., Mazumder medicinal plants against ESbL producing multidrug-resistant A., Pommier Y., (-)-Arctigenin as a lead structure for inhibitors enteric bacteria, Microbiological Research, 2007, 162, 264-275. of human immunodeficiency virus type-1 integrase, J Med [31] Lin R.D., Chin Y.P., Lee M.H, Antimicrobial activity of Chem., 1996, 39, 86–95. antibiotics in combination with natural flavonoids against [19] Pirvu L., Hlevca C., Nicu I., Bubueanu C., Comparative clinical extended-spectrum beta-lactamase (ESBL)-producing Analytical, Antioxidant and Antimicrobial Activity Studies on Klebsiella pneumoniae, Phytother Res, 2005, 19, 612-617. a Series of Vegetal Extracts Prepared from Eight Plant Species Growing in Romania, Journal of Planar Chromatography, 2014, 27, 346-356. [20] Pirvu L., Nicorescu I., Hlevca C., Albu B., Nicorescu V., Epilobi Hirsuti Herba Extracts Influence the In Vitro Activity of Usual Antibiotics on Standard Bacteria, Open Chemistry, 2016, 14, 1-11.