lucia pirvu*, isabela nicorescu, cristina hlevca, bujor ... · open chem., 2017; 15: 92–102...

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 BacteriaDOI 10.1515/chem-2017-0012received January 23, 2017; accepted March 14, 2017.

Abstract: This work aimed to study the potential effects of four Arctii folium extracts, 5 mg gallic [GAE] acid equivalents per 1 mL sample, on six antibiotics (Ampicillin/AM, Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole-Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/CN) tested on four Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Staphylococcus epidermidis ATCC 12228) and five Gram-negative (Proteus mirabilis ATCC 29245, Escherichia coli ATCC 35218, E. coli ATCC 11229, E. coli ATCC 8739, and Bacillus cereus ATCC 11778) bacteria. Arctii folium extracts were the whole ethanol extract/W and subsequent ethyl acetate/EA, aqueous/AQ, and chloroform/CHL fractions. Chemical qualitative analysis (HPTLC method) emphasized five main polyphenol compounds in Arctii folium polar extracts: chlorogenic acid (Rf≈0.52/0.55) and its isomer, 1,5-di-O-caffeoylquinic acid (Rf≈0.90/0.92), plus cynarin (Rf≈0.77), hyperoside (Rf≈0.68/0.64) and isoquercitrin (Rf≈0.69/0.71). Microbiological screening indicated Arctii folium polar extracts (AQ and W) efficacy on S. epidermidis ATCC 12228; the MIC values were in the range of common antibiotics, being 32 and 128 μg GAE per mL sample respectively. The unpredictable effects (stimulatory or

inhibitory) of Arctii folium extracts in combination with typical antibiotics as well as a potential use of the whole ethanol extract/W for restoring the antimicrobial potency of susceptible antibiotics have also been evidenced.

Keywords: burdock leaves, interaction with usual antibiotics, stimulatory and inhibitory effects

1 IntroductionArctium lappa L. (Asteraceae family), commonly greater burdock, is a biennial species found across most of tEurope, Asia and also America. The root part, Bardanae radix, is used as a herbal medicine, but is also used as a vegetable in Eastern Asian regions (similar to potatoes) due to its nutritional value [1], the high content of carbohydrates (69%) including inulin (27-50%) and mucilage added to fats, proteins and numerous vitamins (B1, B2, B3, B5, B6, B9, C, E and K) and minerals (Ca, Fe, Mg, Mn, P, K, Na and Zn) [2] respectively.

Concerning the basis of its medicinal use, data indicate that Bardanae radix is rich in caffeoylquinic acid isomers [1, 3], with dicaffeoylquinic acid derivatives being quantified at 75.4% and 1,5-di-O-caffeoyl-4-O-maloylquinic 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, essential oils, guainolides and bitter compounds, lignans (e.g., arctigenin, arctiin,) and sterols (e.g., sitosterol, stigmasterol) [2]. Studies regarding the metabolic profile of the compounds of burdock plant pieces indicated caffeoylquinic acid derivates and luteolin and quercetin rhamnosides occur in the root part, caffeoylquinic acid and quercetin, quercitrin and luteolin derivates in the leaves, whereas cynarin and chlorogenic acid are dominant in the seeds [5].

*Corresponding author: Lucia Pirvu: National Institute of Chemical-Pharmaceutical Research and Development (ICCF), Vitan 112, 031299,Bucharest, Romania, E-mail: [email protected] Isabela Nicorescu: Institute of Hygiene and Veterinary Public Health, Campul Mosilor 5, 021201, Bucharest, Romania Cristina Hlevca, Bujor Albu: National Institute of Chemical-Pharmaceutical Research and Development (ICCF), Vitan 112, 031299,Bucharest, Romania Valentin Nicorescu: University of Agronomic Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Splaiul Independentei 105, 050097, Bucharest, Romania

© 2017 Lucia Pirvu et al., published by De Gruyter Open.This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Unauthenticated

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Burdock Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Antibiotics on Bacteria 93

Based on this valuable phytochemical content, Bardanae radix represents an important ingredient for traditional medicine practitioners, and has also achieved international recognition for its numerous healthy effects [1]; Romanian folk medicine also considers a bardanae radix hot water extract (decoction type) 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 burdock products and corresponding separate extracts or specific compounds (especially lignans such as arctiin and arctigenin, and polyphenols compounds) having antioxidant, anti-inflammatory, antitumor, antibacterial and antiviral activities [1], as well as hepatoprotective [7], antiurolithiasic [8] and skin improving [1, 9] effects, with some clinical studies also proving their effectiveness in knee osteoarthritis [10] and for dental infectious conditions [11, 12]; burdock root and fruits are also reported to have antidiabetic activity [1, 4], with their beneficial effects being attributed to the copresence of sitosterol, inulin and lignans[2]. Furthermore, the combination arctigenin - polyphenols have shown the capacity to block tumor cell growth and metastasis [13]. Additionally, arctiin, caffeic and chlorogenic acid mixtures have proven significant anti-mutagenic effects and a positive correlation with polyphenolic content [14]. Concerning the biochemical pathway involved, studies on arctigenin tested on MH60 cells indicated the stimulation of apoptosis (IC50 = 1.0 μM) as being the most probable mechanism [15], while the capacity of polyphenols to protect the cells against radical oxygen species (ROS) injuries and to stimulate immune processes [16] are believed to effectively contribute to the antitumor effect of burdock products.

The leaves of Arctium lappa L. have been recognized as havingsome antimicrobial properties against some bacterial strains found in endodontic infections such as Bacillus subtilis, Lactobacillus acidophilus and Pseudomonas aeruginosa, as well as on Candida albicans, Escherichia coli, Staphylococcus aureus and Micrococcus luteus [12]. Furthermore, the efficacy of burdock leaf derived products against influenza A virus (A/NWS/33, H1N1) (IFV) [17], herpesvirus (HSV-1, HSV-2), adenovirus (ADV-3, ADV-11), and human immunodeficiency virus (HIV-1) [18] has also been proven.

Thus, in the current upward trend of using plant derived products as natural medicines, while at the same time increasing bacterial resistance to antibiotics of all classes, based on these data, but also on our previous results [19, 20] demonstrating the antimicrobial effects of some whole and selective extracts from medicinal plants

growing in Romania, this study was aimed to evaluate any potential interaction of Arctii folium extracts with typical antibiotics. The results of the study are very useful to avoid situations of incompatibility with such antibiotics, but also as the starting point for developing new natural synergistic products able to restore antimicrobic susceptibility.

2 Experimental Procedure

2.1 Materials

2.1.1 Plant material description

Leaves from burdock (Arctii folium) were harvested in June and September from the Romanian Carpathians, Sinaia region (1,000 m altitude). Taxonomic identification was carried out by the botanist’s team of the National Institute of Chemical-Pharmaceutical R&D (ICCF Bucharest), Romania. The burdock leaves were shade dried and ground to a medium-size plant powder; voucher specimens (ALam7.1, Alam7.2) are deposited in the ICCF Plant Material Storing Room.

2.1.2 Vegetal extracts preparation

Fifty (50) g of leaf powder (Arctii folium) were heat-assisted extracted (twice) with 500 mL of 70% (v/v) ethanol. The total extract obtained was filtered through a (medium) paper filter, resulting in 650 mL ethanol extract (E7). Two hundred and fifty (250) mL of (E7) ethanol extract was concentrated at low pressure at sicc product then passed into 20% (v/v) propylene glycol (20% PG) solution so as to ensure a final concentration of exactly 5 mg total phenols (expressed as gallic acid [GAE] equivalents) per 1 mL extract, further called Arctii folium whole ethanol extract (codified W); the whole ethanol extract (W) was divided into 2 mL Eppendorf tubes and stored at -8 °C until microbiological studies. Another two hundred and fifty (250) mL of (E7) ethanol extract was also concentrated at sicc product then dissolved into 100 mL of distilled water. The resulting aqueous solution was (manually) extracted first with (3 x 100 mL) chloroform and then with (3 x 100 mL) ethyl acetate solvent, 24 hours per each stage. Three selective extracts were obtained: aqueous (AQ), ethyl acetate (EA) and chloroform (CHL) fractions, respectively. The three Arctii folium selective fractions were analyzed as concerning total phenols content (GAE per 1 mL sample) then concentrated at sicc product and (separately) passed

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94 Lucia Pirvu et al.

into 20% PG as follows: aqueous and ethyl acetate fractions were prepared so as to achieve identical 5 mg GAE / 1 mL samples, while the chloroform fraction (CHL) was passed into 20% PG by means of the whole extract algorithm so as to assure the same amount of non-polar compounds. Similarly, the three selective extracts, AQ, EA and CHL (test extracts) were divided into monodoses and stored at -8 °C until microbiological studies.

2.1.3 Chemicals, reagents and references

Reagents (Folin-Ciocalteau and Natural Product) and solvents (methanol, ethanol, ethyl acetate, formic acid, glacial acetic acid and chloroform) were purchased from Sigma-Aldrich Co, Fluka and Biochemika, as well as quercetin (95%), rutin (min. 95%), quercitrin (>90%), isoquercitrin (>90%), hyperoside (>97%), apigenin (>97%), vitexin-2’’O-rhamnoside (>98%), apigenin 7-O-apiosylglucoside/apiin (>99%), chlorogenic acid (>95%), rosmarinic acid (97%), caffeic acid (>98%) and cynarin (analytical standard) polyphenols compounds – the reference substances (ref.).

2.2 Experimental Design

2.2.1 Qualitative (HP)TLC analysis

Studies were performed using TLCand a Linomat 5, CAMAG apparatus, (Muttenz – Switzerland) according to a general method for the assessment of polyphenols (Wagner and Bladt [21] and Reich and Schibli [22]), as described in previous studies [19, 20].

2.2.2 Estimation of Total Phenolics Content

Studies were performed according to the Folin-Ciocalteu method [23], also detailed in previous studies [19, 20].

2.2.3 Antimicrobial Activity Assay

2.2.3.1 Test organisms Nine reference bacterial strains (ATCC strains purchased from Thermo Scientific Romania) were used, including four Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Staphylococcus epidermidis ATCC 12228) and five Gram-negative bacteria (Proteus mirabilis

ATCC 29245, Escherichia coli ATCC 35218, E. coli ATCC 11229, E. coli ATCC 8739, and Bacillus cereus ATCC 11778). Nutrient broths (Oxoid CM0001) and Muller-Hinton agar (Oxoid CM0337) culture media were used (prepared from a dehydrated base according with the manufacturer’s recommendations).

2.2.3.2 Test antimicrobialsSix antibiotics (microtablets purchased from Biolab Zrt. Romania) were used: Ampicillin (AM, 10 μg), Gentamicin (CN, 10 μg), Tetracycline (TE, 30 μg), Sulfamethoxazole/ Trimethoprim (SXT, 25 μg), Ciprofloxacin (CIP, 5 μg), and Chloramphenicol (C, 30 μg).

2.2.3.3 Inoculum preparation The microorganisms were stored in freezing conditions and activated by cultivation in Nutrient broth at 37 °C for 24 hours. Bacterial inoculums were prepared from an overnight broth culture in demineralised water in order to obtain a bacterial turbidity equivalent to 0.5 McFarland standards [24].

2.2.3.4 Minimum inhibitory concentration (MIC) assayThe experiments were accomplished using the reference method for testing the in vitro activity of the antimicrobial agents, ISO 20776-1/2006 [25] and CLSI M07-A9 document [26]. MIC appraisals were carried out using an accurate working scheme (see Table 1S) as follows: four identical Arctii folium dilution series were prepared, corresponding to four test extracts, i.e. the whole ethanol extract/W 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 by incorporation of each vegetal extract into Muller-Hinton Agar (MHA) medium so that the concentration level of the four test extracts (dilution series) ranged in 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% solution (4.1 ml + 0.9 ml) was used as the negative control. After the solidification of the MHA mixed with the tested vegetal extract, the bacterial inoculum (104 cfu/spot) was applied to the surface of the medium and the plates were incubated for 24 hours at 37 °C. The results were interpreted according to literature data [27] by evaluating the bacterial growth at respective vegetal extract dilution series and concentration of phenols.



2.2.3.5 Synergism assay The assessment of potential interactions between the four Arctii folium extracts and the six antibiotics (microtablets of accurate concentration) has as scientific basis and further design study model their combination at the MIC/2 concentration level [28]. Thus, the culture medium (MHA) was prepared by incorporating Arctii folium extracts so 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 1 – chromatograms A1, A2 and A3) indicated the three polar extracts (the whole ethanol extract/W, ethyl acetate fraction/EA and aqueous fraction/AQ) as containing two main polyphenol subclasses, the caffeoylquinic acid isomers (blue fluorescent/fl. spots - s2, s3’, s5’ and s6) and quercetin derivates (orange fl. spots - s1, s3, s4 and s5) and the nonpolar, chloroform fraction/CHL, which did not reveal any polyphenol compounds. Based on the literature data [5], but also on the current reference substances/ref. used (see T4, T5, T6, T7 and T8 tracks), the main spots were attributed to rutin (s1/Rf≈0.43), chlorogenic acid (s2/Rf≈0.52/0.55 – the major compound in the whole ethanol extract/W and subsequent aqueous fraction/AQ), neochlorogenic acid (s3’/Rf≈0.61/0.64) hyperoside (s3/

Rf≈0.68/0.64), isoquercitrin (s4/Rf≈0.69/0.71), quercitrin (s5/Rf≈0.77), cynarin (s5’/Rf≈0.77) and another major caffeoylquinic acid isomer (s6/Rf≈0.90/0.92 – the major compound in ethyl acetate fraction/EA) attributed to 1,5-di-O-caffeoyl-4-O-maloylquinic acid [4].

Furthermore, some differences were revealed between the extracts obtained from leaves collected at different

Figure 1: TLC aspects of Arctium lappa folium extracts comparatively to the reference substances (ref.). A1: Track T1, quercetin-3-O-rutinoside/ rutin, chlorogenic acid, apigenin-7-O-apiosyl-glucoside/ apiin, quercetin-3-O-galactoside/ hyperoside, rosmarinic acid, and apigenin (ref); Tracks T2, burdock leaves whole (70%, v/v) ethanol extract/W – duplicate sample; Track T3, rutin, vitexin-2’’-O-rhamnoside, luteolin-7-O-glucoside/ cynaroside, quercetin-3-O-rhamnoside/ quercitrin, and quercetin (ref.); A2: Tracks T2aq, burdock leaves aqueous fraction/AQ – duplicate sample; Track T2ea, burdock leaves ethyl acetate fraction/EA – duplicate sample; Track T2chl, burdock leaves chloroform fraction/CHL – single sample. A3: Track T4, rutin, chlorogenic acid, caffeic acid (ref); Track T5, quercetin-3-O-glucoside/ isoquercitrin (ref); Track T6, quercetin-3-O-galactoside/ hyperoside (ref); Track T2ea, burdock leaves ethyl acetate fraction/EA; Track T2aq, burdock leaves aqueous fraction/AQ; Track T2, burdock leaves whole ethanol extract/W; Track T7, cynarin (ref). Track T8; caffeic acid (ref).



vegetation times; Arctii folium derived products from September (chromatogram A3) did not reveal the presence of quercetin-3-O-rutinoside/rutin and corresponding monoglycoside, quercetin-3-O-rhamnoside/quercitrin, previously seen [19] in Arctii folium extracts during the June period (chromatograms A1 and A2).

Regarding chemical quantitative aspects, the whole ethanol extract/W, and aqueous/AQ and ethyl acetate/EA selective extracts were made to provide the exact content of 5 mg total phenols (GAE) / mL sample.

Designed so as to ensure identical levels of the non-polar compounds as the whole extract/W, the selective chloroform extract/CHL also indicated small amounts of phenolic compounds (0.125 mg GAE / mL sample), even if not confirmed by TLC studies.

3.2 Evaluation of the minimum inhibitory concentration (MIC)

Defined as 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 (CLSI, ISO), minimum inhibitory concentration (MIC) appraisal was carried out using an accurate working scheme (Table 1S) and precisely quantified test vegetal samples as concerning the total phenols content (μg GAE per 1 mL sample).

The results (Table 1) suggest reserved antimicrobial potency of Arctii folium extracts, the best results being shown on S. epidermidis ATCC 12228 (precisely the aqueous

fraction/AQ and the whole extract/W), with MIC values measuring 32 and 128 μg GAE / mL sample.

On P. mirabilis ATCC 29245 and S. aureus ATCC 25923 as well as on S. aureus ATCC 6538, the whole extract/W and the aqueous fraction/AQ indicated identical MIC values of 256 μg GAE / mL sample.

The results also reveal the lack of activity of all tested extracts against B. cereus ATCC 11778, E. faecalis ATCC 29212 and the three E. coli ATCC strains, as well as the inefficiency of the nonpolar chloroform fraction (CHL) on all microbial strains studied.

Furthermore, the nine bacterial strains properly developed on MHA and MHA plus (20%, v/v) propylene glycol solution (MHA + PG) medium, thus proving the lack of influence on the bacterial growth (positive control).

3.3 Effects of Arctii folium extracts upon (six) antibiotics

Studies have been done on six antibiotics (Ampicillin/AM, Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole-Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/CN), each one belonging to a separate class of antibiotic, with the antimicrobial used being classed by the official reports of the European Union as effective [29], and in the same time being extensively used for treating infections worldwide.

As a working principle, all combinations (antibacterial substance - vegetal extract) were studied on all bacterial strains (Table 2), excepting those combinations and strains

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

No. crt.

Bacterial strain MIC (μg GAE / mL) Control sample (MHA + PG)

Whole extract (W)

Aqueous fraction (AQ)

Ethyl acetate fraction (EA)

Cloroform 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



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).



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. aureus 6538

S. aureus25923

S.epidermidis12228

P. mirabilis 29245

E. coli 35218

E. coli 11229

E.coli 8739 B.cereus11778

E.faecalis29212

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.



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 control/C), Ampicillin/AM (Figure 2) and Arctii folium extracts showed the best results against E. coli ATCC 11229 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 folium extracts demonstrated stimulatory, but inhibitory, eff ects too. The most important stimulatory eff ects were provided by the whole extract (W) in case of B. cereus ATCC 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 non-polar extracts on all tested bacteria, excepting S. epidermitis ATCC 12228 and E. coli ATCC 8739; the most important synergistic activities were obtained against E. faecalis ATCC 29212 (induced by polar extracts), both

Figure 2: Bacterial strains response to Ampicillin/AM combinations with the four Arctii folium extracts (AM+W, AM+AQ, AM+EA, and AM+CHL) comparatively to the antibiotic alone, AM (C); diameter of the inhibition zone, (mm) ± SD.

Figure 3: Bacterial strains response to Tetracycline/TE combinations with the four Arctii folium extracts (TE+W, TE+AQ, TE+EA, and TE+CHL) comparatively to the antibiotic alone, TE (C); diameter of the inhibition zone, (mm) ± SD.

Figure 4: Bacterial strains response to Cyprofl oxacin/CIP combinations with the four Arctii folium extracts (CIP+W, CIP+AQ, CIP+EA, and CIP+CHL) comparatively to the antibiotic alone, CIP (C); diameter of the inhibition zone, (mm) ± SD.



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 extract/W were E. faecalis 29212, followed by the two S. aureus strains, P. mirabilis ATCC 29245 and B. cereus ATCC 11778; an important inhibitory eff ect of the aqueous 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% and 31% stimulatory eff ects compared to the antibiotic alone therefore appraised as 12% and 10% synergistic activity; Sulfamethoxazole-Trimethoprim/SXT led to 25% 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 inhibitory eff ects or no eff ects; the only exception was that of Tetracycline/TE which showed stimulatory eff ects when combined with the EA fraction. It must be noticed that Arctii folium polar extracts (AQ and W) indicated a

Figure 5: Bacterial strains response to Sulfamethoxazole-Trimethoprim/SXT combinations with the four Arctii folium extracts (SXT+W, SXT+AQ, SXT+EA, and SXT+CHL) comparatively to the antibiotic alone, SXT (C); diameter of the inhibition zone, (mm) ± SD.

Figure 6: Bacterial strains response to Chloramphenicol/C combinations with the four Arctii folium extracts (C+W, C+AQ, C+EA, and C+CHL) comparatively to the antibiotic alone, C (C); diameter of the inhibition zone, (mm) ± SD.

Figure 7: Bacterial strains response to Gentamicin/CN combinations with the four Arctii folium extracts (CN+W, CN+AQ, CN+EA, and CN+CHL) comparatively to the antibiotic alone, CN (C); diameter of the inhibition zone, (mm) ± SD.



certain antimicrobial activity on S. epidermidis 12228 (MIC values measuring 32 and 128 μg GAE / mL sample), so that the lack of synergistic activity of Arctii folium extracts along with antibiotics tested convey the normal behavior of a synergistic product.

The P. mirabilis ATCC 29245 strain (Figure 3S) proved to be sensitive especially to the whole extract/W combination with SXT (the stimulatory effect was appraised up to 40% compared to the antibiotic alone meaning 17% synergistic activity), but also with CIP (up to 20% stimulatory effect compared to the antibiotic meaning 7% synergistic activity) and CN (up to 28% stimulatory effect compared to the 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 sensitivity after combining TE, C and AM with Arctii folium all polar extracts (W, AQ and EA): TE combinations led

Table 3. General view of the effects of Arctii folium extracts on the six antibiotics tested.

Type of effect Antibiotic tested / number of cases

AM TE CIP SXT C CN

Lack of interaction (meaning identical Ø and ND situations)

15 10 11 16 11 15

Inhibitory effect 4 1 10 10 4 7

Stimulatory effect 17 25 15 10 21 14

Stimulatory effect over 5 mm Ø (synergistic effects)

12 16 6 3 13 6

Figure 8. The situation upon specific antibiotic a) and individual extract b).



to 89%, 67%, and 111% antimicrobial efficacy increases compared to the antibiotic alone meaning 21%, 7%, and 36% synergistic activities; C combinations led up to 42%, 47%, and 47% antimicrobial efficacy boosts and 12%, 17%, and 17% synergistic activities, while AM combinations lead up to 32%, 42%, and 37% antimicrobial efficacy boosts and 4%, 12%, and 8% synergistic activities respectively.

Table 3 and Figure 8 (a and b) cumulate the results obtained by testing the 24 pairs of chemical–vegetal antimicrobial combinations on the nine microbes.

Thus, with 22 synergistic interactions (meaning significant biological activity certified by a combined inhibition zone exceeding 5 mm diameter) and only one situation of inhibitory effect, Arctii folium whole (70%, v/v) ethanol extract/W with exactly 5 mg gallic [GAE] acid equivalents per 1 mL sample appear as the most realistic synergistic product along with the six (susceptible) antimicrobials tested, mainly with the Tetracycline/TE antibiotic.

4 ConclusionsTherefore, based on the present study, three conclusions and potential uses have been drawn: first of all, the unpredictable effect (stimulatory or inhibitory) of Arctii folium derived products (mainly the aqueous extract/AQ) in combination with current antibiotics has been demonstrated; secondly, the results suggest potential uses of Arctii folium whole (70%, v/v) ethanol extract in restoring the activity of the antibiotics affected by microbial resistance; lastly (and also backed up by our previous studies on Epilobi hirsuti herba extracts [20]), further studies on separate compounds and flavonols (eg., myricetin and quercetin derivates [31]) combinations with phenylcarboxilic acids (e.g., gallic and caffeic acid derivates) are proposed as a starting point for developing new, natural synergistic agents useful in the antibiotic industry. Found to be present in all of the polar extracts (W, AQ and EA), the two main quercetin monoglycosides, hyperoside (s3) and isoquercitrin (s4) respectively, are the most probable active (synergistic) compounds in Arctii folium derived products. While the quantity and rate of these two quercetin monoglycosides, as well as the co-presence of different quantity and quality of phenolic acids could explain the similarities, but also the differences between the three polar vegetal extracts, the microbial susceptibility to antibiotics and vegetal compounds could explain the final reaction of bacteria to each combination studied. Regarding the non-polar, chloroform fraction

(CHL), this could be active on the basis of its non-polar status thus allowing a better internalization of the active compounds into bacteria.

Finally, the results may be useful for physicians and pharmacists who are currently using phytomedicines and plant based medicines, in order to avoid potential interactions between Arctii folium derived products and antibiotics.

Acknowledgements: The authors from National Institute of Chemical Pharmaceutical Research and Development (ICCF) - Bucharest Romania, gratefully acknowledge the financial support from project POC ID P_40_406, SMIS 105542 sustaining the design and development of plant derived products, their chemical characterization and data processing.

Conflict of interest: The authors declare no competing financial interest.

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lucia pirvu*, isabela nicorescu, cristina hlevca, bujor ... · open chem., 2017; 15: 92–102...

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