2. Taraxacum extracts versus commercial antibiotics

Strains Scedosporium

QC 7870

Serratia marcescens

Serratia/Rahnella

Staphylococcus S. aureus (MRSA) 43,300

S. aureus (MSSA) 25,923

S. aureus NCTC 6571

Staphylococcus

NCTC 14990

T. longifusus

T. 4193

V. albo-atrum V. albo-atrum F-2437 V. cholera ATCC 11623

V. 2471

V. 2471

X. campestris VKM

Table 1.

Principal types of bioassays carried out to determine the

608

 Broth dilution assay

 IC50 (50% growth

inhibition)

antimicrobial

 activity of the genus

Taraxacum and their respective results.

parahaemolyticus

 KCTC

Disc diffusion method.

Broth dilution.

parahaemolyticus

 KCTC

Disc diffusion method.

Inhibition zone. %

Control (8 mm). None.

 500–2000 μg/mL

 9.5–15 mm.

[21]

5.1–97.9%

Inhibition

Inhibition zone. %

Control (8 mm). None.

 500–2000 μg/mL

6–10 μM

 9.5 - 15 mm.

[23]

84.0–97%

1.0–1.2 μM

 [5]

Inhibition

Broth Inhibition method

 Agar diffusion method

Microtiterd

 method

 IC56

 MIC

None Erythromicin

Cefixime 1.0 μM

 1.0 μM.

27 mg/mL

15.6–250 μg/mL

 No activity

12.5 μM

 [11]

[8]

Broth dilution assay

 IC50, MIC,

None

15 μM

>15 μM; >15 μM;

[10]

Morphological

changes

mentagrophytes

 UPCC

Agar well diffusion

 Inhibition zone,

Canesten (55 mm, 4.3)

 30 μg

12 mm, 0.2

 [9]

antimicotic

 index

Agar well diffusion, agar

No information

 No information

> tube dilution

 epidermidis

 sp.

 Disk diffusion method

Diet

 Disk diffusion method

 Disk diffusion method

 Disk diffusion method Disk diffusion method

Disk diffusion method

 Inhibition zone

 Inhibition zone

CFU count

 Inhibition zone

 Inhibition zone

 Inhibition zone

 Inhibition zone

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

No information

 Inhibition

[45]

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

Control

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL No information

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

 >200 mg/mL

 >200 mg/mL

 Inhibition

 >200 mg/mL

 >200 mg/mL

 >200 mg/mL

 >200 mg/mL

 [12]

 [12]

 [12]

 [12]

 [12]

[8]

 [12]

apiospermum

Disk diffusion method

 Inhibition zone

Ciprofloxacin

 5 μg/disc

 130–200 mg/mL

 >200 mg/mL

 [12]

Bioassay

Results expresion

 Positive control

Active

concentration

 Main results

 Reference

284 Herbal Medicine

When comparing Taraxacum extracts to commercial antibiotics, C. jejuni adhesion was controlled by a Taraxacum extract with an IC50 value of 2.7 mg/mL, slightly less compared to the 3.4 mg/mL obtained with 3<sup>0</sup> -sialyllactose [28]. In another study, a T. officinale extract showed MIC values of 0.004 mg/mL, similar to chloramphenicol with MIC values of 0.001–0.06 mg/mL but considerably lower than amphotericin B with MIC values of 0.4 –0.8 μg/mL for different Gram positive and Gram negative bacteria, respectively [13]. The MIC value of 1.0 mg/mL for M. luteus was similar for a methanolic extract and for erythromycin and cefixime, but considerably lower than the MIC value of 12.5 mg/mL obtained for V. cholera [8]. In the same work, the inhibition percentage for Aspergillus spp. and Rhizoctonia spp. was 37–84%, relatively lower than terbinafine at 12 mg/mL and 100% inhibition.

Generally, researchers select only one technique for evaluating the antimicrobial performance of Taraxacum. Few studies have assessed agar disc diffusion and broth dilution in parallel, even when the limitations and advantages for both bioassays have been already stated, as indicated above. An example of this includes the antibacterial properties of an ethanolic extract of the T. mongolicum flower, whose fractions were examined by both bioassays [19]. The authors indicated that at 0.1 mg/disc, inhibition results were relatively lower for the plant extract compared to gentamicin and tetracycline, with values between 7.12 and 19.4 mm for the plant extracts and 18.9–38.8 mm for the antibiotics. However, MIC values of 0.06–0.5 mg/mL were obtained for plant extracts against the tested strains. Antibiotics had much lower MIC values of 3.0–5.0 μg/mL, which reaffirms the fact that different bioassays need to be performed in parallel to accurately evaluate the antimicrobial effectiveness of an extract.

and root extract of T. officinale at 150–200 mg/mL was inactive against 24 bacterial strains, but

*Taraxacum* Genus: Extract Experimental Approaches http://dx.doi.org/10.5772/intechopen.72849 287

The conclusion of these studies may be misleading if slight dilutions or excessively high concentrations are tested. For example, experiments with quantities higher than 1.0 mg/mL for extracts or 0.1 mg/mL for isolated pure compounds should be avoided, whereas the presence of activity is very interesting when concentrations are below 0.1 μg/mL for extracts, and 0.01 mg/mL for isolated compounds [1]. Even when promising results have been achieved, the extracts have also shown contradictory results and can mislead the actual potential of this

In general, active concentrations of Taraxacum extracts that achieve inhibitions similar to the synthetic antibiotics are 100–10,000 times higher, which makes Taraxacum extracts unsuitable for pharmaceutical development at the moment. However, this is expected since synthetic antibiotics are pure, concentrated compounds, whereas plant extracts are a mixture of different, dilute compounds that act synergistically or antagonistically. Because this situation is common and a characteristic of plant extracts, some authors indicate the possibility of using antibiotics synergistically with plant extracts to improve the action mechanisms against antibiotic-resistant bacteria. No research regarding the synergistic use of Taraxacum genus has yet been performed [47]. At present, only commercial and synthetic antibiotics, such as kanamycin, amphotericin B, terbinafine, chloramphenicol, and cephalothin, among others, have been considered as positive controls for establishing strain sensitivity. Comparisons of Taraxacum with natural, commercially available antibiotic compounds (such as propolis and other honey products) have been neglected: only one study, regarding antibacterial agents for dental care, contains a comparison with propolis [41]. The comparison with natural antibiotics, for example, honey, might be more realistic in traditional medicine due to the similar vegetable origin and characteristics. As long as no pure compound extraction or purification of Taraxacum extracts can be performed reliably for testing antimicrobial activity, the real potential of the Taraxacum genus

Alternatively, instead of only utilizing a chemical antibiotic or a natural antibiotic, antimicrobial synergistic interactions between plant bioactives and some common antibiotics have been reported. There are many advantages to using antimicrobial compounds from medicinal plants, such as fewer side effects, better patient tolerance, lower expense, acceptance due to

Regarding the expression of the results, the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and inhibition percentage of growth are cited by researchers as the most common measurements of antimicrobial performance. In this sense, there are two primary categories for measuring an antimicrobial agent: bactericidal or bacteriostatic. Bacteriostatic refers to an agent that prevents the growth of bacteria and a bactericidal agent kills bacteria, but a complete separation of these definitions might be further pursued. This difference only applies under strict laboratory conditions and is inconsistent for a

ciprofloxacin at 5.0 μg/disc showed high antimicrobial activity [12].

plant extract if no further investigation is pursued.

as a source of natural therapeutic agents cannot be established.

3. Expression of results in antimicrobial studies

long history of use, and renewability [48].

The weak activity that some authors have indicated could be improved by higher concentrations, which are needed to reach quantifiable antimicrobial activity under different conditions and assays. For instance, concentrations of T. officinale extracts at 130–500 mg/ mL were needed to achieve the effect of amphotericin B at 0.2–0.4 μg/mL against Candida strains [26]. In the cases of mancozeb, carboxin, thiram, and benomyl, only 1 mg/disc was effective in inhibiting the growth of R. solani, F. oxysporum, and C. sativus, while the Taraxacum extract needed a concentration of 5 mg/disc to achieve the same effect [31]. For H. pylori and C. jejuni, growth was inhibited by ampicillin and gentamicin at concentrations of 0.5–5.0 μg/mL, while an extract of 500 mg/mL was needed to achieve this inhibition [29]. Considering the disc assay method, an extract of ethyl acetate at 10 mg/mL showed minor inhibition zones (14–18 mm) against A. hydrophila, S. typhi, S. aureus, B. cereus, and E. coli as compared to cephalothin at 0.03 mg/mL (18–24 mm) [9]. In this study, inhibition diameters were only 20–25% smaller than those reached by the synthetic antibiotic, but the extract concentration was more than 300 times higher, as well as 100 times higher than what would normally be indicated for an attractive natural antibiotic in a commercial setting. In a similar study, the inhibition zones of chloramphenicol at 0.02 mg/mL (10.7–23.5 mm) against E. coli and S. aureus were lower compared to an ethanolic extract of T. officinale at 200 mg/mL (25–30 mm) [36]. In this case, the extract showed higher activity but its concentration was 10,000 times higher than its respective antibiotic. Moreover, methanolic extracts of T. officinale at 50 mg/mL resulted in inhibition similar to tetracycline at 0.1 mg/mL using broth dilution and disc assay methods against E. coli, S. aureus, and B. subtilis, among others; that is, a concentration 500 times greater than the antibiotic was necessary to obtain a similar effect [4].

In several studies, different Taraxacum extracts exhibited no activity under the tested conditions. For instance, embedded discs with 50 μL of an ethanolic extract of T. officinale were not active compared to controls, such as ticarcillin, at 75 μg/disc, and chloramphenicol, at 30 μg/ disc [26]. Another study, using a similar extract at 2.5 mg/disc, was inactive against certain strains, as compared to gentamycin, at 1.0 mg/disc, and tetracycline, at 2.0 mg/disc [16]. Different extracts of T. officinale leaves and roots (chloroform, methanol, and water) were not active towards Mycobacterium compared to streptomycin at 1.14 μg/mL [43]. An ethanolic extract of T. phaleratum was also inactive against the same strain compared to rifampin at 0.005–0.01 μg/mL, isoniazid at 0.05–0.1 μg/mL, and kanamycin at 2.5–5.0 μg/mL [44]. A leaf and root extract of T. officinale at 150–200 mg/mL was inactive against 24 bacterial strains, but ciprofloxacin at 5.0 μg/disc showed high antimicrobial activity [12].

Generally, researchers select only one technique for evaluating the antimicrobial performance of Taraxacum. Few studies have assessed agar disc diffusion and broth dilution in parallel, even when the limitations and advantages for both bioassays have been already stated, as indicated above. An example of this includes the antibacterial properties of an ethanolic extract of the T. mongolicum flower, whose fractions were examined by both bioassays [19]. The authors indicated that at 0.1 mg/disc, inhibition results were relatively lower for the plant extract compared to gentamicin and tetracycline, with values between 7.12 and 19.4 mm for the plant extracts and 18.9–38.8 mm for the antibiotics. However, MIC values of 0.06–0.5 mg/mL were obtained for plant extracts against the tested strains. Antibiotics had much lower MIC values of 3.0–5.0 μg/mL, which reaffirms the fact that different bioassays need to be performed in parallel to accurately evaluate the antimicrobial effectiveness of an

The weak activity that some authors have indicated could be improved by higher concentrations, which are needed to reach quantifiable antimicrobial activity under different conditions and assays. For instance, concentrations of T. officinale extracts at 130–500 mg/ mL were needed to achieve the effect of amphotericin B at 0.2–0.4 μg/mL against Candida strains [26]. In the cases of mancozeb, carboxin, thiram, and benomyl, only 1 mg/disc was effective in inhibiting the growth of R. solani, F. oxysporum, and C. sativus, while the Taraxacum extract needed a concentration of 5 mg/disc to achieve the same effect [31]. For H. pylori and C. jejuni, growth was inhibited by ampicillin and gentamicin at concentrations of 0.5–5.0 μg/mL, while an extract of 500 mg/mL was needed to achieve this inhibition [29]. Considering the disc assay method, an extract of ethyl acetate at 10 mg/mL showed minor inhibition zones (14–18 mm) against A. hydrophila, S. typhi, S. aureus, B. cereus, and E. coli as compared to cephalothin at 0.03 mg/mL (18–24 mm) [9]. In this study, inhibition diameters were only 20–25% smaller than those reached by the synthetic antibiotic, but the extract concentration was more than 300 times higher, as well as 100 times higher than what would normally be indicated for an attractive natural antibiotic in a commercial setting. In a similar study, the inhibition zones of chloramphenicol at 0.02 mg/mL (10.7–23.5 mm) against E. coli and S. aureus were lower compared to an ethanolic extract of T. officinale at 200 mg/mL (25–30 mm) [36]. In this case, the extract showed higher activity but its concentration was 10,000 times higher than its respective antibiotic. Moreover, methanolic extracts of T. officinale at 50 mg/mL resulted in inhibition similar to tetracycline at 0.1 mg/mL using broth dilution and disc assay methods against E. coli, S. aureus, and B. subtilis, among others; that is, a concentration 500 times greater than the antibiotic was necessary to obtain

In several studies, different Taraxacum extracts exhibited no activity under the tested conditions. For instance, embedded discs with 50 μL of an ethanolic extract of T. officinale were not active compared to controls, such as ticarcillin, at 75 μg/disc, and chloramphenicol, at 30 μg/ disc [26]. Another study, using a similar extract at 2.5 mg/disc, was inactive against certain strains, as compared to gentamycin, at 1.0 mg/disc, and tetracycline, at 2.0 mg/disc [16]. Different extracts of T. officinale leaves and roots (chloroform, methanol, and water) were not active towards Mycobacterium compared to streptomycin at 1.14 μg/mL [43]. An ethanolic extract of T. phaleratum was also inactive against the same strain compared to rifampin at 0.005–0.01 μg/mL, isoniazid at 0.05–0.1 μg/mL, and kanamycin at 2.5–5.0 μg/mL [44]. A leaf

extract.

286 Herbal Medicine

a similar effect [4].

The conclusion of these studies may be misleading if slight dilutions or excessively high concentrations are tested. For example, experiments with quantities higher than 1.0 mg/mL for extracts or 0.1 mg/mL for isolated pure compounds should be avoided, whereas the presence of activity is very interesting when concentrations are below 0.1 μg/mL for extracts, and 0.01 mg/mL for isolated compounds [1]. Even when promising results have been achieved, the extracts have also shown contradictory results and can mislead the actual potential of this plant extract if no further investigation is pursued.

In general, active concentrations of Taraxacum extracts that achieve inhibitions similar to the synthetic antibiotics are 100–10,000 times higher, which makes Taraxacum extracts unsuitable for pharmaceutical development at the moment. However, this is expected since synthetic antibiotics are pure, concentrated compounds, whereas plant extracts are a mixture of different, dilute compounds that act synergistically or antagonistically. Because this situation is common and a characteristic of plant extracts, some authors indicate the possibility of using antibiotics synergistically with plant extracts to improve the action mechanisms against antibiotic-resistant bacteria. No research regarding the synergistic use of Taraxacum genus has yet been performed [47].

At present, only commercial and synthetic antibiotics, such as kanamycin, amphotericin B, terbinafine, chloramphenicol, and cephalothin, among others, have been considered as positive controls for establishing strain sensitivity. Comparisons of Taraxacum with natural, commercially available antibiotic compounds (such as propolis and other honey products) have been neglected: only one study, regarding antibacterial agents for dental care, contains a comparison with propolis [41]. The comparison with natural antibiotics, for example, honey, might be more realistic in traditional medicine due to the similar vegetable origin and characteristics. As long as no pure compound extraction or purification of Taraxacum extracts can be performed reliably for testing antimicrobial activity, the real potential of the Taraxacum genus as a source of natural therapeutic agents cannot be established.

Alternatively, instead of only utilizing a chemical antibiotic or a natural antibiotic, antimicrobial synergistic interactions between plant bioactives and some common antibiotics have been reported. There are many advantages to using antimicrobial compounds from medicinal plants, such as fewer side effects, better patient tolerance, lower expense, acceptance due to long history of use, and renewability [48].
