Taraxacum Genus: Extract Experimental Approaches

Abstract

This chapter presents factors or considerations to be taken into account when selecting the procedure or method for obtaining extracts and bioactive compounds. The genus Taraxacum has proved to have several interesting properties and there are numerous techniques and bioassays used to test the antimicrobial properties of extracts. However, the extraction process is crucial to optimize the final biological outcomes. Extraction procedures that until now have been used are simple and inexpensive, however, we wanted to report a series of studies that group valuable results, which could be useful for future studies, enhancing the research carried out by authors from all over the world and also allowing the interrelated study of this genus.

1. Introduction

Taraxacum has been worldwide tested against several bacterial and fungal strains under various extract conditions and bioassays, and we compiled enough published information with the aim of comparing and/or relationship between the various existing methods and their result in the antimicrobial profile.

1.1. Antimicrobial bioassay methods used in Taraxacum genus

Several different methods have been used for testing antimicrobial activity, the application of which most often depends on the available instrumentation and the training of the investigators [1]. Screening for antibacterial and antifungal activity is often done by agar disc diffusion, agar well test diffusion, and agar dilution or microdilution broth. In agar disc diffusion, a paper disc soaked with the extract is laid on top of an inoculated agar plate and is generally used as a preliminary check for antibacterial activity prior to more detailed studies. In the agar well test diffusion method, the extract is deposited into wells cut into the agar and can be used as a screening method when large numbers of extracts or large numbers of bacterial isolates are to be screened. In the agar dilution method, a known concentration of the extract is mixed with the agar prior to strain inoculation. In some cases, the inoculated plates or tubes are exposed to UV light to screen the presence of light-sensitizing photochemicals. In the broth dilution method, different techniques exist for determining the end-point, such as an optical density measurement or the enumeration of colonies by viable count. Antimicrobial activity can also be analyzed by a spore germination assay in broth or on glass slides. In situ antifungal activity can be achieved by electron microscopy techniques such as scanning and transmission, as well as by confocal laser scanning microscopy [2].

Direct tissue inoculation is the least used testing method, probably due to the inherent characteristics of the substrate (fruits, vegetables, etc.) that can affect the final results and the standardized laboratory conditions needed for proper result comparisons. Authors also indicate certain restrictions regarding the use of a specific technique. For instance, diffusion techniques seem to be inadequate for non-polar extracts, although many reports with these techniques have been published. Furthermore, when only a small amount of sample is available, diffusion techniques can be considered more appropriate [3]. The disc diffusion method is quick and easy but has several serious shortcomings, such as false positives and negative results due to poor test substance solubility and diffusion through the semi-solid nutritive medium [1].

The agar diffusion and microdilution broth methods are the two most common techniques for determining the antimicrobial activities of Taraxacum extracts, but the results are not always reproducible; factors, such as the volume and concentration of the extract placed on the paper disc and the solvent used, vary considerably between studies. When results are compared, the different sensitivities of the assays make antimicrobial activity highly dependent on the selection of the proper test. For example, aqueous fractions of T. officinale showed no activity in the disc diffusion test but moderate toxicity against E. coli and B. subtilis in the broth dilution test [4]. Considering this issue, a list of the bioassays used for testing Taraxacum extracts against every strain identified, including the main results, is presented in Table 1.

Strains	Bioassay	Results expresion	Positive control	Active concentration	Main results	Reference
Alternaria alternata	Broth dilution assay	IC50, MIC, Morphological changes	None	15 μM	2.9 μM; 1.0 μM; +	[5]
A. alternata	Paper disc diffusion method	% Inhibition	None	S, S/2, S/10, S/100	16.7–76.2%	[6]
A. carbonarius (Bainier) Thom	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	4%, 0%	[7]
A. flavus 0064	Agar tube dilution	Inhibition growth	Terbinafine 12 mg/mL 100%	15 mg/mL	70.80%	[8]
A. fumigatus 66	Agar tube dilution	Inhibition growth	Terbinafine 12 mg/mL 100%	16 mg/mL	84.80%	[8]
A. hidrophila (food poisoning patients)	Agar diffusion method	Inhibition zone	Cephalotin 30 μg/mL (20 mm)	10 mg/mL	No activity	[9]
A. niger	Broth dilution assay	IC50, MIC, Morphological changes	None	15 μM	4.2 μM; 2.8 μM; +	[5]
A. niger 0198	Agar tube dilution	Inhibition growth	Terbinafine 12 mg/mL 100%	17 mg/mL	37.40%	[8]
A. niger UPCC 3701	Agar well diffusion	Inhibition zone, antimicotic index	Canesten (23 mm, 1.3)	30 μg	No activity	[9]
A. niger van Thiegem	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	3%, 45%	[7]
A. niger VKM F-33	Broth dilution assay	IC50 (50% growth inhibition)	None	6–10 μM	1.2–5.6 μM	[10]
A. niger VKM F-33	Microtiterd method	IC50	None	15.6–250 μg/mL	No activity	[11]
A. flavus QC 6658	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
A. fumigatus	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
A. niger	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
B. cereus	Agar diffusion method	Inhibition zone	Cephalotin 30 μg/mL (22 mm)	10 mg/mL	18 mm	[9]
B. cereus (spoiled rice)	Agar diffusion method	Inhibition zone	Cephalotin 30 μg/mL (20 mm)	10 mg/mL	18 mm	[9]
B. cereus ATCC 1778	Broth dilution assay	MIC	Cloramphenicol (0.004 μM)	No information	2.5 μM	[13]
B. cereus NCTC 7464	Broth dilution assay	MIC	None	2 mg/mL	500 μg/mL	[14]
B. cereus NCTC 7464	Microtiterd method	MIC	None	2 mg/mL	250 μg/mL	[15]
B. cinérea	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	9%, 38%	[7]
B. cinerea SGR-1	Broth dilution assay	IC50 (50% growth inhibition)	None	6–10 μM	5.2–5.8 μM	[5]
B. cinerea SGR-1	Microtiterd method	IC51	None	15.6–250 μg/mL	No activity	[11]
B. sorokiniana	Broth dilution assay	IC50, MIC, Morphological changes	None	15 μM	>15 μM; >15 μM;	[10]
B. sorokiniana 6/10	Broth dilution assay	IC50 (50% growth inhibition)	None	6–10 μM	5.2 μM	[5]
B. sorokiniana F-1446	Microtiterd method	IC52	None	15.6–250 μg/mL	No activity	[10, 11]
B. subtilis	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	weak activity, not indicated	[16]
B. subtilis	Agar inoculation	MIC	None	No information	7.0 mg/mL	[17]
B. subtilis	Disc diffusion method	Inhibition zone	None	No information	12.04 mm	[18]
B. subtilis	Disc diffusion method	Inhibition zone	Tetracyclin 10 μg/disc (23.6 mm)	10 mg/mL	5.1–97.9%	[19]
B. subtilis	Broth dilution	MIC	Tetracyclin (MIC 5.0 μg/mL)	10 μg/mL	5.1–97.9%	[19]
B. subtilis	Agar well diffusion	Inhibition zone	None	120 μg/mL	10.0–14.0 mm	[20]
B. subtilis	Broth dilution assay	MIC	Tetracyclin 100 μg/mL (84%)	50 mg/mL	10–54%	[4]
B. subtilis	Disc diffusion method	Inhibition zone	Tetracyclin 100 μg/mL (22 mm)	50 mg/mL	11–19 mm	[4]
B. subtilis ATCC 1149	Agar well diffusion	Inhibition zone, antimicotic index	Chloramphenicol (8 mm, 0.3)	30 μg	11 mm, 0.1	[9]
B. subtilis ATCC 6633	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM	18 mg/mL	No activity	[8]
B. subtilis ATCC 6633	Disc diffusion method	Inhibition zone	Control (8 mm)	1000–2000 μg/mL	0–12.5 mm	[21]
B. subtilis ATCC 6633	Broth inhibition method	% Inhibition	None	1000–2000 μg/mL	5.1–97.9%	[21]
B. subtilis ATCC 6633	Broth dilution assay	MIC	None	No information	No activity	[22]
B. subtilis KCTC 1021	Disc diffusion method	Inhibition zone	Control (8 mm)	500–2000 μg/mL	8.5–12.5 mm	[23]
B. subtilis KCTC 1021	Broth dilution	% Inhibition	None	1000–2000 μg/mL	5.1–97.9%	[23]
B. subtilis VKM 1053	Agar diffusion method	Inhibition zone		6–10 μM	0.8–1.2 μM	[5]
B. cereus NCTC 7464	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
B. pumilus (wildtype hand isolate)	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
B. subtilis NCTC 10400 (NCIMB 8054)	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
C. albicans	Disc diffusion method	Inhibition zone	No information	No information	No activity	[24]
C. albicans	Broth dilution assay	MIC	Tetracyclin 100 μg/mL (68%)	50 mg/mL	0–70%	[4]
C. albicans	Disc diffusion method	Inhibition zone	Tetracyclin 100 μg/mL (20 mm)	50 mg/mL	14–20 mm	[4]
C. albicans ATCC 10231	Paper disc diffusion method	Inhibition zone	Chloramphenicol 30 mcg (27 mm)	50 μL/disc	No activity	[25]
C. albicans ATCC 10231	Agar diffusion method	Inhibition zone	Anfotericin (0.2 mm)	200 μg/mL	>200 μg/mL	[26]
C. albicans ATCC 18804	Broth dilution assay	MIC	Anfotericin B (0.0004 μM)	No information	0.039 μM	[13]
C. albicans ATCC 90028	Agar well diffusion		Anfotericin B (100 μg/disc)	40 μg	3.0 mm	[27]
C. albicans UPCC 2168	Agar well diffusion	Inhibition zone, antimicotic index	Canesten (18 mm, 0.3)	30 μg	12 mm, 0.2	[9]
C. glabrata ATCC 2001	Agar diffusion method	Inhibition zone	Anfotericin (0.4 mm)	200 μg/mL	>200 μg/mL	[26]
C. gloesporoides	Broth dilution assay	IC50, MIC, Morphological changes		15 μM	>15 μM; >15 μM;-	[10]
C. jejuni	Broth dilution assay	Adhesion, cytotoxicity, Antibacterial	3-sialyllactose (IC50 1.4 mg/mL)	500 mg/mL	IC50 < 3 mg/mL, <10%, no activity	[28]
C. jejuni NCTC 11168 (ATCC 700819)	Broth dilution assay	MIC, IC50, % Inhibition	Ampicillin (IC50 1.61 μg/mL)	15 μM	No activity	[29]
C. lagenarium	Direct inoculation	Rates of Inhibition	Control untreated leaves	No information	1.90	[30]
C. lagenarium	Direct inoculation	Rates of Inhibition	Control untreated leaves	No information	12.80	[30]
C. neoformans ATCC 32608	Broth dilution assay	MIC	Anfotericin B (0.0008 μM)	No information	0.039 μM	[13]
C. parapsilepsis ATCC 22019	Agar diffusion method	Inhibition zone	Anfotericin (0.4 mm)	200 μg/mL	>200 μg/mL	[26]
C. sativus (S. Ito and Kurib.)	Paper disc diffusion method	Inhibition zone	Mancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)	5 mg/disc	weak activity, not indicated	[31]
C. tropicalis ATCC 750	Agar diffusion method	Inhibition zone	Anfotericin (0.4 mm)	200 μg/mL	2.0 mm	[26]
C. utilis ATCC 22023	Agar diffusion method	Inhibition zone	Anfotericin (0.4 mm)	200 μg/mL	>200 μg/mL	[26]
C. violaceum ATCC 12472	Quorum sensing	Inhibition zone	None	No information	7 mm	[32]
C. violaceum ATCC 31532	Quorum sensing	Inhibition zone	None	No information	No activity	[33]
C. violaceum NTCT 13274	Quorum sensing	Inhibition zone	None	No information	No activity	[33]
C. albicans	Disk diffusion method	Inhibition zone	Ciprofloxacin (5 μg/disc)	130–200 mg/mL	>200 mg/mL	[12]
C. glabrata ATCC 2001	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
C. krusei ATCC 6258	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
C. parapsilosis ATCC 22019	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
C. michiganense subesp. Michiganense Ac-1144	Agar diffusion method	Inhibition zone		6–10 μM	0.8–1.4	[5]
Cupriavidus sp.	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
E. coccus ATCC 13048	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM	19 mg/mL	No activity	[8]
E. coli	Disc diffusion, broth dilution	Inhibition zone, MIC	Erythromicin (MIC 27 μg/mL)	10–500 μg/mL	13.3 mm, MIC 50 μg/mL	[34]
E. coli	Agar inoculation	MIC	None	No information	1.0 mg/mL	[17]
E. coli	Agar diffusion method	Inhibition zone	None	0.1–1.0 mg/mL	>0.5 mg/mL (1–4 mm)	[35]
E. coli	Agar diffusion method	Inhibition zone	Cloramphenicol 10 mg/mL (30.5 mm)	50–200 mg/mL	5.25–23.5 mm	[36]
E. coli	Diet	CFU count	Control	No information	Inhibition	[8]
E. coli	Disc diffusion method	Inhibition zone	None	1 g/mL	10.2–18.5 mm	[37]
E. coli	Disc diffusion method	Inhibition zone	None	No information	13.21 mm	[18]
E. coli	Disc diffusion method	Inhibition zone	Gentamycin 10 μg/disc (18.9 mm)	10 mg/mL	12.05–14.21 mm	[19]
E. coli	Broth dilution	MIC	Gentamycin (MIC 1.25 μg/mL)	10 μg/mL	250–500 μg/mL	[19]
E. coli	Disc diffusion method	Inhibition zone	No information	No information	11–13 mm	[24]
E. coli	Agar well diffusion	Inhibition zone	None	120 μg/mL	2.0–3.0 mm	[20]
E. coli	Broth dilution assay	MIC	Tetracyclin 100 μg/mL (78%)	50 mg/mL	14–62%	[4]
E. coli	Disc diffusion method	Inhibition zone	Tetracyclin 100 μg/mL (18 mm)	50 mg/mL	12–15 mm	[4]
E. coli 7075	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
E. coli 8739	Agar diffusion method	Inhibition zone	None	1 mg/mL	>1 mg/mL	[38]
E. coli ATCC 11229	Disc diffusion method	Inhibition zone	Control (8 mm)	500–2000 μg/mL	11–13.5 mm	[21]
E. coli ATCC 11229	Broth inhibition method	% Inhibition	None	500–2000 μg/mL	98.1–100%	[21]
E. coli ATCC 1229	Broth dilution assay	MIC	None	No information	No activity	[22]
E. coli ATCC 15224	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM	20 mg/mL	No activity	[8]
E. coli ATCC 25322	Agar well diffusion	Inhibition zone	Gentamycin	240 mg/mL	6.5 mm	[39]
E. coli ATCC 8677	Paper disc diffusion method	Inhibition zone	Ticarcillin 75 mcg (27 mm)	50 μL/disc	No activity	[26]
E. coli ATCC 8739	Broth dilution assay	MIC	None	S, S/2	Inhibition	[40]
E. coli DSM 1103	Broth dilution assay	MIC	None	2 mg/mL	No activity	[14]
E. coli DSM 1103	Microtiterd method	MIC	None	2 mg/mL	No activity	[15]
E. coli KCTC 2441	Disc diffusion method	Inhibition zone	Control (8 mm)	500–2000 μg/mL	9.0–12 mm	[23]
E. coli KCTC 2441	Broth dilution	% Inhibition	None	1500–2000 μg/mL	13–98.4%	[23]
E. coli NCTC 25922	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
E. coli NCTC 9001	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
E. coli UPCC 1195	Agar well diffusion	Inhibition zone, antimicotic index	Chloramphenicol (25 mm, 3.2)	30 μg	11 mm, 0.1	[9]
E. faecalis	Irrigation in situ		None	7 mg/mL	weak activity, not indicated	[41]
E. faecalis ATCC 19433	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM. Cefixime 1.0 μM	21 mg/mL	No activity	[8]
E. coli 0157 NCTC 12900	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
E. coli DH5	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Enterobacter/Klebsiella sp.	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
E. faecalis NCTC 775	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Exophiala (Wangiella) dermatitidis QC 7895	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
F. avenaceum	Broth dilution assay	IC50, MIC, Morphological changes		15 μM	13.1 μM; 6.7 μM; +	[10]
F. graminearum VKM F-1668	Broth dilution assay	IC50 (50% growth inhibition)		6–10 μM	>10 μM	[5]
F. oxysporium Schlecht	Paper disc diffusion method	Inhibition zone	Mancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)	6 μM	5.7 μM	[31]
F. oxysporium TSKHA-4	Broth dilution assay	IC50 (50% growth inhibiton)	Kanamycin	6 μM	5.7 μM	[5]
F. oxysporium TSKHA-4	Microtiterd method	IC53	None	15.6–250 μg/mL	No activity	[11]
H. pylori	Paper disc diffusion method	Inhibition zone	Control (8 mm)	No information	10 mm	[42]
H. pylori NCTC 11639 (ATCC 43629)	Broth dilution assay	MIC, IC50, % Inhibition	Gentamicin (IC50 0.081 μg/mL)	500 mg/mL	25%	[29]
K. pneumoniae	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
K. pneumoniae	Agar diffusion method	Inhibition zone	Cloramphenicol 10 mg/mL (26.5 mm)	50–200 mg/mL	—	[36]
K. pneumoniae	Disc diffusion method	Inhibition zone	Gentamycin 10 μg/disc (18.9 mm)	10 mg/mL	13.24–17.72 mm	[19]
K. pneumoniae	Broth dilution	MIC	Gentamycin (MIC 5.0 μg/mL)	10 μg/mL	125–250 μg/mL	[19]
K. pneumoniae ATCC 13866	Broth dilution assay	MIC	Cloramphenicol (0.001 μM)	No information	0.625 μM	[13]
K. pneumoniae UC 5	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM. Cefixime 1.0 μM	22 mg/mL	No activity	[8]
K. aerogenes NCTC 9528	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
K. pneumoniae 700,603	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
L. monocytogenes KCCM 40307	Disc diffusion method. Broth Inhibition method	Inhibition zone. % Inhibition	Control (8 mm). None	500–2000 μg/mL	0–12 mm. 5.1–97.9%	[21]
L. monocytogenes KCCM 40307	Disc diffusion method. Broth Inhibition method	Inhibition zone. % Inhibition	Control (8 mm). None	500–2000 μg/mL	10.5–12 mm. 27.2–94%	[23]
L. monocytogenes NCTC 11994	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
M. aureum 4721 E	Broth dilution	MIC	Streptomycin (IC50 1.14 μg/mL)	500 μg/mL	>500 μg/mL	[41]
M. bovis BCG	Broth dilution	MIC	Streptomycin (IC50 1.14 μg/mL)	500 μg/mL	>500 μg/mL	[43]
M. canis	Agar well diffusion, agar tube dilution	No information	No information	No information	Inhibition	[44]
M. kristinae	Agar inoculation	MIC	None	No information	5.0–7.0 mg/mL	[17]
M. laxa	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	11%, 5%	[7]
M. luteus	Agar well diffusion	Inhibition zone	None	120 μg/mL	5.0–9.0 mm	[20]
M. luteus ATCC 10240	Agar diffusion method	MIC	Erythromicin 1.0 μM	23 mg/mL	1.0 μM	[8]
M. piriformis	Paper disc diffusion method	% Inhibition	None	S, S/2, S/10, S/100	5.3–66.7%	[6]
M. smegmatis MC2 155	Broth dilution	MIC	Streptomycin (IC50 1.14 μg/mL)	120 μg/mL	5.3–66.7%	[43]
M. tuberulosis H37RA	Broth dilution method	MIC	Rifampim. Isoniazid. Kanamycin	No information	> 200 μg/mL	[44]
MRSA (clinical isolated)	Broth dilution assay. Microtiterd method	MIC	None	2 mg/mL	375 μg/mL. 500 μg/mL	[14]
MRSA AARM 3696	Disc diffusion method	Inhibition zone	Gentamicin 0.2 mg/disc (20.1 mm)	0.5–3.0 mg/disc	6.8–16.5 mm	[45]
P. acnes	Broth dilution assay	No information	No information	No information	No information	[46]
P. aeruginosa	Broth dilution assay	No information	No information	No information	No information	[46]
P. aeruginosa	Disc diffusion, broth dilution	Inhibition zone, MIC	Erythromicin	10–500 μg/mL	No activity	[34]
P. aeruginosa	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
P. aeruginosa	Agar diffusion method	Inhibition zone	Cloramphenicol 10 mg/mL (26.0 mm)	50–200 mg/mL	—	[36]
P. aeruginosa	Disc diffusion method	Inhibition zone	Gentamycin 10 μg/disc (20.0 mm)	10 mg/mL	16.52–19.19 mm	[19]
P. aeruginosa	Broth dilution	MIC	Gentamycin (MIC 2.5 μg/mL)	10 μg/mL	125–250 μg/mL	[19]
P. aeruginosa	Agar well diffusion	Inhibition zone	None	120 μg/mL	8.0–13.0 mm	[20]
P. aeruginosa ATCC 15442	Broth dilution assay	MIC	Cloramphenicol (0.015 μM)	No information	2.5 μM	[13]
P. aeruginosa ATCC 7221	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM. Cefixime 1.0 μM	24 mg/mL	No activity	[8]
P. aeruginosa ATCC 9027	Broth dilution assay	MIC	None	No information	No activity	[22]
P. aeruginosa ATCC 9721	Paper disc diffusion method	Inhibition zone	Ticarcillin 75 mcg (20 mm)	50 μL/disc	No activity	[25]
P. aeruginosa UPCC 1244	Agar well diffusion	Inhibition zone, antimicotic index	Chloramphenicol (23 mm, 2.8)	30 μg	11 mm, 0.1	[9]
P. betae	Broth dilution assay	IC50, MIC, Morphological changes		15 μM	10.7 μM; 8.0 μM; +	[10]
P. betae F-2532	Microtiterd method	IC54	None	15.6–250 μg/mL	No activity	[11]
P. debaryanum VKM F-1505	Broth dilution assay	IC50 (50% growth inhibition)	None	6–10 μM	2.6 μM	[5]
P. digitatum (Perss) Sacc	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	12%, 42%	[7]
P. expansum	Paper disc diffusion method	% Inhibition	None	S, S/2, S/10, S/100	15.7–69.7%	[6]
P. expansum Link.	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	No activity	[7]
P. expansum Link. ATCC 42710	Paper disc diffusion method	Inhibition zone	Control (8 mm)	0.1 g/mL	12 mm	[47]
P. infestans	Microtiterd method	IC55	None	15.6–250 μg/mL	No activity	[11]
P. infestans OSV 12	Direct inoculation (potato disc)	Disease development	None	1.3–5.3 μM	1.3–5.2 μM	[5]
P. italicum Wehmer	Microassay method on slides	ICG, IGTE	Only in vivo assays (Imazalil, Fenhexamid)	0.75X	2%, 56%	[7]
P. mirabilis	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
P. mirabilis	Agar diffusion method	Inhibition zone	None	0.1–1.0 mg/mL	>0.5 mg/mL (4–10 mm)	[35]
P. ovale	Broth dilution assay	No information	No information	No information	No information	[46]
P. syringeae VKM B-1546	Agar diffusion method	Inhibition zone		6–10 μM	1.2–1.3 μM	[5]
P. vulgaris	Disc diffusion, broth dilution	Inhibition zone, MIC	Erythromicin (MIC 26 μg/mL)	10–500 μg/mL	10.1 mm, MIC 100 μg/mL	[34]
P. vulgaris	Agar inoculation	MIC	None	No information	5.0 mg/mL	[17]
P. vulgaris	Disc diffusion method	Inhibition zone	Gentamycin 10 μg/disc (19.5 mm)	10 mg/mL	13.38–18.33 mm	[19]
P. vulgaris	Broth dilution	MIC	Gentamycin (MIC 2.5 μg/mL)	10 μg/mL	250–500 μg/mL	[19]
Penicillium sp. QC 743275	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Pseudomona sp.	Disc diffusion method	Inhibition zone	No information	No information	11–21 mm	[24]
P. aeruginosa NCTC 1662	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
P. aeruginosa NCTC 27853	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Pseudomonas sp.	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
R. solani 18,619	Agar tube dilution	Inhibition growth	Terbinafine 12 mg/mL 100%	25 mg/mL	77.47%	[8]
R. solani Kühn	Paper disc diffusion method	Inhibition zone	Mancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)	5 mg/disc	4.75–17.63 mm	[31]
S. aureus NCTC 8178	Microtiterd method	MIC	None	2 mg/mL	500 μg/mL	[15]
S. abony enterica NCTC 6017	Broth dilution assay	MIC	None	S, S/2	Inhibition	[40]
S. agalactiae	Disc diffusion method	Inhibition zone	None	1 g/mL	11.1–19.7 mm	[37]
S. aureus	Broth dilution assay	No information	No information	No information	No information	[46]
S. aureus	Disc diffusion, broth dilution	Inhibition zone, MIC	Erythromicin (MIC 33 μg/mL)	10–500 μg/mL	12.7 mm, MIC 50 μg/mL	[34]
S. aureus	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
S. aureus	Agar inoculation	MIC	None	No information	5.0 mg/mL	[17]
S. aureus	Agar diffusion method	Inhibition zone	None	0.1–1.0 mg/mL	>0.5 mg/mL (1–4 mm)	[35]
S. aureus	Agar diffusion method	Inhibition zone	Cloramphenicol 10 mg/mL (35.0 mm)	100–200 mg/mL	9.0–10.75 mm	[36]
S. aureus	Disc diffusion method	Inhibition zone	None	1 g/mL	9.7–19.9 mm	[37]
S. aureus	Disc diffusion method	Inhibition zone	None	No information	16.15 mm	[18]
S. aureus	Disc diffusion method	Inhibition zone	Tetracyclin 10 μg/disc (38.8 mm)	10 mg/mL	11.22–15.07 mm	[19]
S. aureus	Broth dilution	MIC	Tetracyclin (MIC 2.5 μg/mL)	10 μg/mL	250 μg/mL	[19]
S. aureus	Disc diffusion method	Inhibition zone	No information	No information	11–21 mm	[24]
S. aureus	Agar well diffusion, agar tube dilution	No information	No information	No information	Inhibition	[44]
S. aureus	Agar well diffusion	Inhibition zone	None	120 μg/mL	4.0–11.0 mm	[20]
S. aureus	Broth dilution assay	MIC	Tetracyclin 100 μg/mL (75%)	50 mg/mL	0–76%	[4]
S. aureus	Disc diffusion method	Inhibition zone	Tetracyclin 100 μg/mL (17 mm)	50 mg/mL	9–18 mm	[4]
S. aureus (salted white cheese)	Agar diffusion method	Inhibition zone	Cephalotin 30 μg/mL (24 mm)	10 mg/mL	16 mm	[9]
S. aureus ATCC 12600	Paper disc diffusion method	Inhibition zone	Chloramphenicol 30 mcg (27 mm)	50 μl/disc	No activity	[25]
S. aureus ATCC 25922	Agar well diffusion	Inhibition zone	Penicillin	320 mg/mL	10.4 mm	[39]
S. aureus ATCC 43300	Disc diffusion method	Inhibition zone	Erythromycin 50 μg/well	40 μg	7.5 mm	[27]
S. aureus ATCC 6530	Broth dilution assay	MIC	None	No information	No activity	[22]
S. aureus ATCC 6538	Broth dilution assay	MIC	Cloramphenicol (0.063 μM)	No information	5.0 μM	[13]
S. aureus ATCC 6538	Broth dilution assay	MIC	None	S, S/2	No activity	[40]
S. aureus ATCC 6538	Agar diffusion method	Inhibition zone, MIC	Erythromicin 1.0 μM. Cefixime 1.0 μM	26 mg/mL	No activity	[8]
S. aureus ATCC 6538	Disc diffusion method	Inhibition zone	Control (8 mm)	1500–2000 μg/mL	0–12 mm	[22]
S. aureus ATCC 6538	Broth inhibition method	% Inhibition	None	1500–2000 μg/mL	5.1–97.9%	[22]
S. aureus ATCC 6538	Agar diffusion method	Inhibition zone	None	1 mg/mL	>1 mg/mL	[38]
S. aureus KCTC 1916	Disc diffussion method	Inhibition zone	Control (8 mm)	500–2000 μg/mL	8.5–13.5 mm	[24]
S. aureus KCTC 1916	Broth dilution	% Inhibition	None	500–2000 μg/mL	98.1–100%	[24]
S. aureus KCTC 3881	Disc diffussion method	Inhibition zone	Gentamicin 0.2 mg/disc (20.1 mm)	0.5–3.0 mg/disc	6.8–16.5 mm	[45]
S. aureus NCTC 8178	Broth dilution assay	MIC	None	2 mg/mL	375 μg/mL	[14]
S. aureus UPCC 1143	Agar well diffussion	Inhibition zone, antimicotic index	Chloramphenicol (20 mm, 2.3)	30 μg	No activity	[9]
S. australis	Broth dilution assay	Growth	None	10–10,000 ppm	No activity	[46]
S. cerviseae	Broth dilution assay	MIC	Tetracyclin 100 μg/mL (50%)	50 mg/mL	0–64%	[4]
S. cerviseae	Disc diffussion method	Inhibition zone	Tetracyclin 100 μg/mL 18 mm)	50 mg/mL	12–15 mm	[4]
S. dysgalactiae	Disc diffusion method	Inhibition zone	None	1 g/mL	13.8–9.6 mm	[37]
S. enterica sorovar typhimurium ATCC 13311	Broth dilution assay	MIC	Cloramphenicol (0.001 μM)	No information	5.0 μM	[13]
S. enteriditis	Disc diffussion method	Inhibition zone	No information	No information	No activity	[24]
S. epidermis KCTC 1917	Disc diffussion method	Inhibition zone	Gentamicin 0.2 mg/disc (24.4 mm)	130–200 mg/mL	7.3–16.7 mm	[45]
S. fiexneri	Disc diffusion, broth dilution	Inhibition zone, MIC	Erythromicin	10–500 μg/mL	No activity	[34]
S. haemolyticus	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	Weak activity, not indicated	[16]
S. marscens	Agar inoculation	MIC	None	No information	1.0–5.0 mg/mL	[17]
S. sonnei ATCC 11060	Broth dilution assay	MIC	Cloramphenicol (0.001 μM)	No information	2.5 μM	[13]
S. tiphimurium SARB 69	Broth dilution assay	MIC	None	2 mg/mL	No activity	[14]
S. typhi	Agar well diffussion, agar tube dilution	No information	No information	No information	Inhibition	[44]
S. typhi (food poisoning patients)	Agar diffusion method	Inhibition zone	Cephalotin 30 μg/mL (18 mm)	10 mg/mL	14 mm	[9]
S. typhi H.	Agar diffusion method	Inhibition zone	Gentamicyn 1 mg/disc, Tetracyclin 2 mg/disc	4–12 μg/disc	No activity	[16]
S. typhimurium Reference collection B-69	Microtiterd method	MIC	None	2 mg/mL	No activity	[15]
Salmonella poona NCTC 4840	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Scedosporium apiospermum QC 7870	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Serratia marcescens	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Serratia/Rahnella sp.	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Staphylococcus	Diet	CFU count	Control	No information	Inhibition	[8]
S. aureus (MRSA) 43,300	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
S. aureus (MSSA) 25,923	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
S. aureus NCTC 6571	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
Staphylococcus epidermidis NCTC 14990	Disk diffusion method	Inhibition zone	Ciprofloxacin 5 μg/disc	130–200 mg/mL	>200 mg/mL	[12]
T. longifusus	Agar well diffusion, agar tube dilution	No information	No information	No information	Inhibition	[45]
T. mentagrophytes UPCC 4193	Agar well diffusion	Inhibition zone, antimicotic index	Canesten (55 mm, 4.3)	30 μg	12 mm, 0.2	[9]
V. albo-atrum	Broth dilution assay	IC50, MIC, Morphological changes	None	15 μM	>15 μM; >15 μM; −	[10]
V. albo-atrum F-2437	Microtiterd method	IC56	None	15.6–250 μg/mL	No activity	[11]
V. cholera ATCC 11623	Agar diffusion method	MIC	Erythromicin 1.0 μM. Cefixime 1.0 μM	27 mg/mL	12.5 μM	[8]
V. parahaemolyticus KCTC 2471	Disc diffusion method. Broth Inhibition method	Inhibition zone. % Inhibition	Control (8 mm). None.	500–2000 μg/mL	9.5–15 mm.; 5.1–97.9%	[21]
V. parahaemolyticus KCTC 2471	Disc diffusion method. Broth dilution.	Inhibition zone. % Inhibition	Control (8 mm). None.	500–2000 μg/mL	9.5 - 15 mm.; 84.0–97%	[23]
X. campestris VKM −608	Broth dilution assay	IC50 (50% growth inhibition)		6–10 μM	1.0–1.2 μM	[5]

Table 1.

Principal types of bioassays carried out to determine the antimicrobial activity of the genus Taraxacum and their respective results.

2. Taraxacum extracts versus commercial antibiotics

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′-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.

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].

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].

3. Expression of results in antimicrobial studies

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 particular agent against all bacteria; indeed, it can be influenced by growth conditions, bacterial density, test duration, and extent of reduction in bacterial numbers. Furthermore, bacteriostatic activity has been defined as an MBC/MIC ratio of 4, but numerous technical problems and other factors can affect the determination of that ratio and may have an important impact on the interpretation of the in vivo situation. Although MBC and MIC data may provide information on the potential action of antibacterial agents in vitro, it is necessary to combine this information with pharmacokinetic and -dynamic data to provide more meaningful predictions of efficacy in vivo [49]. Considering this information, no pharmacokinetic or -dynamic studies have been conducted involving the Taraxacum genus to date. The majority of the research (not only as an antimicrobial agent, but also as an important medicinal plant) has been performed from a traditional perspective, based on centuries of oral traditions. Only in recent decades has Taraxacum been subjected to a considerable amount of tests, principally due to its anti-inflammatory and anti-carcinogenic properties [50]. The antimicrobial properties of this genus have been widely known, but only very general studies have been performed to date, with information that is difficult to interconnect as the action mechanisms and the specific compounds involved have not yet been elucidated. Nevertheless, all the data gathered here provides a promising case for the advantageous commercial usage of this genus.

Considering this general approach, most of the research regarding Taraxacum indicates MIC values and inhibition percentages measured in relation to area (in solid cultures) or optical density (in broth cultures). The MBC values were not identified in the consulted references. An observation was made that the MIC definition sometimes differed between publications, another obstacle for data comparison. Some MIC definitions are: “the lowest concentration of the tested products that inhibited the development of microorganisms” [40]; “the lowest concentration required to show a marked inhibition of mycobacterial growth at 72 h” [43]; “the lowest concentration of the compound to inhibit the growth of microorganisms” [19]; and “the lowest sample concentration at which no pink color appeared” [15]. This indicates that MIC values are relative to each study and is compounded by the fact that the complete procedure (including extraction process and sample manipulation) is not standardized and varies considerably among the authors. Furthermore, due to the different solubilities and stabilities of the various compounds in the solvent and the sensitivity of the antimicrobial activity assay performed, directly comparing MIC values is difficult and sometimes confusing. As further examples, in three different studies, the authors reported MIC values in the 0.05–5.0 mg range for ethanol, methanol, or water extracts against S. aureus using broth microdilution or agar diffusion method as bioassays [13, 17, 34]. This meant that only MIC values could be used as a comparison against the positive control under the same conditions and may only be considered as an initial screening for further antimicrobial approaches; it cannot provide a reliable comparison between studies. The MIC/MBC ratio might be an option for making antimicrobial activity more independent of assay conditions if similar extraction conditions and sample manipulation have been performed.

4. Scaling up from in vitro to in vivo assays

Scaling up an antimicrobial assay from controlled, in vitro conditions to that of natural, in vivo conditions can be difficult if no proper considerations are taken. For instance, active concentrations for in vitro conditions frequently cannot be reached in vivo because the infecting microorganisms are never exposed to constant concentrations of an antimicrobial agent. Microorganisms in vivo are subject to competition from other microorganisms present in the tissue, so decreased microbial activity might be due to this competition rather than directly related to the antimicrobial activity of the plant extract. Moreover, temperature, pH, and humidity are more difficult to control in an in vivo system. Another issue to consider is that microorganisms in a microtiter plate are in the form of a suspension, whereas bacteria associated with different illnesses naturally form biofilms (organ and tissue infections, dental plaque, etc.), representing an extra challenge for antimicrobial agents [1]. Until now, only studies regarding fruit and vegetable infections have shown a parallel between in vitro and in vivo responses to Taraxacum extracts (Chapter 1; see Section 2.2.2), but studies in animal tissues and organs have not yet been performed directly.

5. Factors affecting antimicrobial activity of extracts

The following sections are referred and discussed in accordance with the information provided in Table 2 (see Chapter 1) and Figure 1. It should be noted that the impact of the parameters mentioned in these sections, except for solvent selection, on the antimicrobial properties of the Taraxacum genus has not yet been studied.

Figure 1.

Reported extraction conditions to achieve positive antimicrobial results.

5.1. Plant material collection

Scientific criteria should be used in the selection of the sample material. To avoid the use of random criteria, the selection of plants should be made from an ethnopharmacological perspective. All the species tested need to be perfectly described and identified, including location, season, date, and time of day harvested. The use of commercial samples should be limited to cases of standardized extracts or defined phytomedicines [3]. The phytochemical composition of Taraxacum (and plants in general) is known to depend on the season in which it is collected, as well as other ecological and climate factors. For example, sesquiterpene lactones are noticeable in the roots, particularly when harvested in the spring [51]. Sterols, which are present in the leaves throughout the year, are highest during the winter months, whereas levels of sitosterol and cycloartenol esters are highest during periods of sunshine [52]. Few authors indicated in which period of the year the plant was harvested, collected, purchased, or the collection site, another factor that could influence the final concentration of compounds in the extract, even when the same extraction conditions are applied. No Taraxacum studies have investigated a possible relationship between harvesting time or collecting site and its antimicrobial properties. Only one study indicated the environmental conditions in which the plant was grown and collected before the antimicrobial assay [27].

5.2. Species identification

Generally, there is a lack of taxonomic identification of the species characterized, mentioned occasionally as Taraxi radix, Taraxi folium, Taraxi herba, Taraxacum spp., or dandelion, especially when researchers use commercial preparations or purchase the plant from local markets [29, 33, 46]. Samples are commonly obtained in the wild, but the lack of proper identification makes the comparison for antimicrobial properties imprecise for determining the actual efficacy of Taraxacum extracts; therefore, only partial conclusions can be pursued and not always extrapolated. For instance, dandelion is used as a common name for several species: khur mang, a name for dandelion in Tibet, can be used for T. officinale, T. mongolicum, T. tibetanum, and T. Sikkimense [53]. As previously stated, environmental conditions affect the tissue composition of the plants, but few reports indicate the corresponding information for further consultation. The importance of proper identification also relates to the risk of toxicity between morphologically similar, but chemically distinct, plants, which is a potential health risk for the communities that harvest medicinal plants in the wild. Only a small portion of the research available mentions proper, expert identification.

5.3. Plant part utilization

Reports indicate that compounds present in Taraxacum vary within parts of the plant, and even though there are common compounds across sections, these concentrations vary as well [54, 55]. A disadvantage that creates further uncertainties when comparing data is that a considerable amount of studies do not indicate which part of the plant was used. In general, aerial parts (leaves, flowers, and seeds), roots, and whole Taraxacum plants have been used in antimicrobial research. Only one Taraxacum study indicated differences between a root extract and a leaf extract, in which the root extract was active against S. aureus and S. typhi. Extracts of plant roots and herbs of different Taraxacum species endemic to Turkey displayed significant activity against M. canis and T. longifusus [44]. Few studies refer to the antimicrobial properties of Taraxacum derivatives. Pseudomona sp., S. aureus, and E. coli were inhibited in a disc diffusion assay, but C. albicans and S. enteriditis were not inhibited by T. officinale honey [24]. The pH of dandelion honey is considered the probable antibacterial component observed against S. aureus [56]. Analyzing the information gathered in this work (also see Table 2), Taraxacum root extracts are less effective at fungal and bacterial inhibition than the aerial parts and seem to be more effective on Gram positive than Gram negative bacteria.

5.4. Sample manipulation

Several authors propose that plants need to be dried and chopped before extraction. This is a consensus among researches due to the necessity of storing samples prior to processing; however, it is a central issue when testing biological activities because bioactive compounds are highly sensitive and react quickly to changes in environmental conditions. These types of changes are common: a sample is stored at room temperature, refrigerated, frozen, or freeze-dried. In rare cases, further sample manipulation has been reported prior to extraction. Specifically, the removal of lipids and proteins with solvents [31] could also affect the compound profile of the extract and the final antimicrobial activity. In one study, a fresh sample was also homogenized before tested [30]. In our research, sample manipulation seems to be just as adequate whether plant parts are dried under the sun or by oven prior to extraction, or used directly as fresh biomass in extract preparation. Due to the possibility that the material used in the extraction may be contaminated, a white control is considered in the activity bioassays, which is the sample not inoculated with the pathogen, to confirm sterility of the stored sample.

5.5. Extraction procedure

Traditional extraction techniques involve solid-liquid extraction with or without high temperatures (maceration, soaking, reflux, etc.), and are characterized by the use of high solvent volumes and long extraction times. These techniques often produce low bioactive extraction yields, low selectivity, and reproducibility can sometimes be compromised. In a common extraction procedure, plant parts are soaked in solvent for extended periods, the slurry is filtered, the filtrate may be centrifuged multiple times for clarification, and the result may be dried under reduced pressure and re-dissolved in alcohol to a determined concentration. Solid-liquid extractions using soaking, maceration, and homogenization are the most used for Taraxacum (although, to a lesser extent, the Soxhlet procedure has been used). Pressurized liquid extraction, subcritical water extraction, and supercritical fluid extraction are presented as novel techniques with important advantages over traditional solvent extraction, such as rapidity, higher yields, and reduced solvent usage. Microwave-assisted extraction and ultrasonic-assisted extraction are pretreatments that can improve the extraction yield by releasing the compounds from the solid matrix [2]. No studies using these techniques have been conducted for the extraction of antibacterial compounds from Taraxacum because maceration, blending, and boiling are the most common extraction procedures for this genus. In one study, the sample was sonically treated prior to extraction but no conclusion regarding the effectiveness of this pretreatment can be pursued [22].

5.6. Relationship between temperature and extraction time

Temperature directly influences both the solubility equilibrium and mass transfer rate of an extraction process. When temperature is increased, the lower viscosity and surface tension of the solvent improves its diffusion inside the solid matrix, achieving a higher yield and extraction rate along with enhanced diffusivity and solubilization results. The primary disadvantages of applying a higher temperature are increased solvent boil-off and reduced effective contact area between solid and liquid phases. A high temperature can also decrease the cell barrier by weakening the integrity of the cell wall and membrane. Furthermore, bioactive compounds may decompose at high temperatures, which require research on the influence of temperature on the overall yield. Temperatures ranging from cold (4°C), room temperature (20–25°C), and solvent boiling point (50–100°C) have been reported for Taraxacum. The majority of the work was conducted in the range of 20–40°C, where the maceration process was proposed and, to a lesser extent, extraction under boiling temperatures has also been indicated (80–100°C, depending on the solvent). Our findings suggest that inhibitory activity is most probable when using a maceration process at mild temperatures (Chapter 1; See Table 2).

Determination of the duration of the extraction process required to extract the bioactive compounds, that is, the minimum time at which equilibrium of solvent concentration between inner and outer cells is reached, is important. Most bioactive compounds are sensitive to elevated temperatures and are susceptible to thermal decomposition outside of the original matrix. The extraction time mentioned in literature for Taraxacum ranged from 5 min for homogenization, 1–3 hours for boiling, and up to 3 weeks for maceration. A clear relationship between extraction time and antimicrobial activity was not observed in the data presented. However, it is possible that the antimicrobial compounds extracted are relatively stable when extracted by maceration at mild temperatures because numerous positive results regarding inhibitory activity were obtained with this process that included times ranging from 4 hours to 5 days.

5.7. Relationship between sample size, solid to solvent ratio, and agitation speed

The particle size of the plant material influences the extraction rate by affecting the total mass transfer area per unit volume, which increases as particle size is reduced. Several authors chopped and ground Taraxacum plant material, but few indicate the mesh grain utilized in extract powder selection. Bioactive compounds are dissolved from the solid matrix into the solvent by a physical process under mass transfer principles and compound solubility. When the amount of extraction solvent is increased, the possibility of the bioactive compounds in the solid matrix coming into contact increases. However, the removal of solute from the solvent requires energy. Therefore, if more solvent than needed is used, there will be a higher energy consumption, needlessly increasing processing costs. In the literature reviewed for Taraxacum, the sample:solvent ratio ranged between 1:1 and 1:40 w/v. In light of the gathered data, this range has no direct impact on antimicrobial activity but certainly affects the economy of the process. Interestingly, most of the positive results have been achieved with ratios of 1:10–1:4.

A higher agitation speed in solid-liquid extraction is preferred, in accordance with mass transfer theory. In this process, the solute moves from inside the solid to the surface through diffusion or capillary action. Once the compound is on the surface, it is recovered by the solvent through convective mass transfer. Agitation rate affects the mass transfer coefficient (kL) and, at higher rates, improves the convective mass transfer rate, which facilitates the extraction process and leads to increases in extraction yields. For Taraxacum, the agitation speed is not usually mentioned in homogenization processes but the most cited value is 170 rpm. Similarly, for the solid:solvent ratio, no direct impact was found in comparisons of different studies.

5.8. Solvents

One critical parameter in extraction procedures is the solvent used for sequestering bioactives from the plant matrix. Extractants that solubilize antimicrobial compounds from plants have been ranked by factors such as biohazard risk and ease of solvent removal from fractions. Methanol was ranked second to methylene dichloride and superior to ethanol and water. Even though acetone was rated the highest, it is one of the least used solvents for bioactive extraction. Ethanol and methanol, in contrast, are both commonly used for initial extraction yet may not demonstrate the greatest sensitivity in yielding antimicrobial chemicals on an initial screening [57]. Solvents used for the extraction of bioactive compounds from plants are selected according to polarity and the compounds they are capable of solubilizing. Different solvents may modify results. Apolar solvents (cyclohexane, hexane, toluene, benzene, ether, chloroform, and ethyl acetate) primarily solubilize alkaloids, terpenoids, coumarins, fatty acids, flavonoids, and terpenoids; polar solvents (acetone, acetonitrile, butanol, propanol, ethanol, methanol, and water) primarily extract flavonols, lectins, alkaloids, quassinoids, flavones, polyphenols, tannins, and saponins [58].

The impact of solvent selection is recognized as extremely critical. For example, the gathered data indicate that growth inhibition on fungal strains can be reached by using ethanolic extracts but not aqueous extracts. Moreover, in the same study, inhibition of Gram positive and Gram negative bacteria using an aqueous extract was indicated but no inhibition was achieved using an acetone extract against the same strains [17]. However, it has also been reported that water extracts led to better activity than ethanolic extracts against acne strains, which can be useful in the skin care field [46]. Alcohol extracts tend to display better activity against bacteria and fungi than water extracts, the latter being generally ineffective. Crude Taraxacum extracts are commonly used in testing antifungal and antibacterial properties [57], but only a few reports involve the fractioning of the crude sample with other solvents to concentrate and isolate potential compounds related to microbial activity [4, 15, 19, 22, 24, 42]. These authors agree that antimicrobial activity decreases as follows: ethyl acetate > dichloromethane ≈ chloroform > butanol ≈ hexane > water. This indicates that the antimicrobial compounds should be extracted according to the solvent polarities, showing effective extractions from solvents with a polarity index ranging from approximately 3.0 to 7.0 instead of too polar or apolar solvents. Data analysis indicates that solvents with low (0–3.0) and high (6.1–9.0) polarities are less active against microorganisms than medium polarity solvents (3.1–6.0). A list of the solvents used in research regarding Taraxacum antimicrobial activity is presented in Tables 3 and 4.

Taraxacum parts mentioned in the text	Number of extracts tested	Positive antimicrobial activity		Negative; antimicrobial activity
Root	51	17	11%	34	43%
Leaves	38	28	18%	10	13%
Flower	13	10	7%	3	4%
Honey	6	4	3%	2	3%
Herb	3	3	2%	0	0%
Aerial	32	24	16%	8	10%
Whole plant	8	0	0%	8	10%
No information	81	66	43%	15	19%
Total	232	152		80

Table 2.

Summary of the antimicrobial results regarding Taraxacum plant parts tested in main studies.

Polarity index	9.0	8.2	7.7	7.4	6.3	6.3	6.2	6.0	5.9	5.6	5.5	5.4	5.1	5.1	4.4	4.1	4.0	3.1	0.0
Genus/solvent	Water	Ethanol 20%	Ethanol 35%	Ethanol 40–- 45%	Ethanol 70– 75%	Methanol 70%	Acetic acid	Ethanol 80%	Methanol 80%	Ethanol 90%	Methanol 90%	Ethanol 96–100%	Methanol 100%	Acetone	Ethylacetate	Chloroform	Dietylether 80%	Dichloromethane	Hexane
Aeromona															—
Alternaria							+
Aspergillus	—						+		+									—
Bacillus	−/+				+	+	+	+				−/+	−/+	+	+	+		+	−/+
Bipolaris							−/+
Botrytis							+		+
Campylobacter			+	+
Candida	—	—				+						−/+	+					+
Chromobacterium	—									+		—
Cochilibus	+
Colletotrichum	+						—
Cupriavidus	—
Clavibacter							+						+
Enterobacter	—												—
Enterococcus	−/+
Escherichia	−/+	+		—	+	+		—	+		—	−/+	−/+	+	+	+		+	—
Exophiala	—
Fusarium	—						−/+						—
Helicobacter				+								+
Klebsiella	—							—					−/+
Listeria	—												+			+
Micrococcus													+	+
Microsporum												+
Monilinia									+
Mycobacterium					—								—
Mucor	+
Penicillium	−/+								−/+								+	+
Pityrosporum												+
Phoma	+						+
Phytophthora	+
Proteus	—				+			—						+
Pseudomona	−/+	—			+		+	—				−/+	−/+			+		+
Salmonella	—											−/+							—
Saprolegnia												—
Pythium	—						+						+
Rhizoctonia	+												+
Saccharomyces						+
Salmonella				—				—					+		—
Scedosporium	—
Serratia	—													+
Shigella	—												+
Staphylococcus	−/+	—		—	+	+		−/+			—	−/+	−/+	+	+	+		−/+	—
Trichophyton												+						+
Vibrio							—						+
Xanthomonas							+

Table 3.

Antimicrobial activity regarding the solvent tested with Taraxacum genus.

(+) Positive antimicrobial activity report. (−) No antimicrobial activity reported. Empty cells indicate no study has been performed so far.

Solvents used in Taraxacum; extracts	Number of extracts tested	Positive antimicrobial activity		Negative antimicrobial activity
Low polarity (0–3.0)	47	22	9%	25	10%
Medium polarity (3.1–6.0)	100	70	28%	30	12%
High polarity (6.1–9.0)	101	38	15%	63	25%
Total	248	130		118

Table 4.

Summary of the antimicrobial results regarding the polarity of the Taraxacum extracts tested in main studies.

6. Perspectives of potential bioassays

As stated above, reports have shown that the antimicrobial potential of different compounds depends not only on the chemical composition of the extract, but also on the targeted microorganism. Further evaluation of the activity of these plants required the study of different conditions. Different parts of the plant (flowers, leaves, stems, etc.), solvent selection (water, alcohol, and organic solvents), extraction procedure (temperature, pH, time, and equipment), bioassay selection (diffusion, dilution, bioautographic methods), and bioassay conditions (volume of inoculum, growth phase, culture medium used, pH of the media, incubation time, and temperature) among others, complicate the comparison of published data.

Studies of the identification and characterization of Taraxacum compounds are generally unrelated to a particular pharmacological property. Therefore, the extraction methods for identifying and quantifying extract compounds differ in sample manipulation:temperature, extraction time, and solvent (among others parameters), indicating that comparisons of the extraction methods utilized in antimicrobial activity assays are typically invalid. This complicates the establishment of a relationship between compounds isolated from Taraxacum parts and antimicrobial activities.

Nevertheless, Taraxacum has been proven effective against most known strains of bacteria, fungi, and protozoa that attack animals and plants through an in vitro or in vivo approach. All studies of Taraxacum extracts against microbes that cause important human diseases (E. coli, S. aureus, and A. niger, among others) were conducted in vitro, while microbes causing foodborne diseases with economic implications (C. lagenarium for cucumber or S. australis for salmonids) were also tested in vivo. For humans, only antimicrobial in vitro assays were conducted primarily due to the ethical issues of clinical trials. Several authors have mentioned that Taraxacum, despite being used as a well-known medicinal plant for centuries, suffers from a lack of in vivo evidence and clinical trials supporting its use [58], which prevents this genus from attracting the possibility of economic development in the pharmacological industry.

Depending on the bioassay selected, diverse extraction conditions should be tested to study the influence of solvents, temperatures, and other parameters that might change outcomes in the extraction process employed. Authors often use non-standardized procedures derived from self-experience combined with bibliographic references, further complicating comparisons between investigations. Even though there are vast amounts of literature on Taraxacum biochemical composition and antimicrobial activity, few isolated compounds can be directly related to this activity because studies do not always identify the accurate active fraction and its associated components. In bioassays, the extract generally used is a mixture of compounds; therefore, there is a strong possibility that the activity may be due to the synergy of the compounds present in the extract and not related to a specific compound. The identification, extraction, and isolation of these active compounds are major areas of research that can be initially pursued to formulate a promising source of Taraxacum antibiotics. The next step is to test these extracts on in vitro and in vivo systems to establish pharmacodynamics and interactions, facilitating the commercial attractiveness of Taraxacum to the pharmaceutical industry.

The bioavailability, pharmacodynamics, and action mechanisms in Taraxacum bioactives have not yet been addressed. Considering that primarily in vitro and, to a much lesser extent, in vivo studies have been conducted using Taraxacum extracts, direct application is the only route that has been considered. If a bioactive compound is going to be suggested as a potential therapeutic agent, other application routes must be tested. Oral ingestion, injection, or inhalation have different characteristics that need to be considered, such as flavor, compound volatility, stability in stomach pH, and possible organ irritation, among others. Therefore, clinical trials are fundamental to evaluating the suitability of Taraxacum extract use in pharmacological approaches.

7. Conclusion

Only a minor fraction of the Taraxacum species has been tested against microorganisms that cause human, animal, and plant diseases. Considering that species can differ in composition due to environmental and genetic characteristics, the evaluated antimicrobial properties could also differ, which means that there is a considerable potential in establishing this genus as a commercial antimicrobial compound. Currently, this genus is considered to have a mild antimicrobial activity compared to other plants, but its worldwide presence and simple cultivation provide an advantage that needs to be assessed more accurately.

Generally, studies do not provide sufficient details concerning the sample manipulation, extraction procedure, or bioassay used, which are necessary for standardization and further statistical comparison. Therefore, despite the published data, it is not possible to conclude which solvent or which conditions provide the optimal results for antimicrobial activity; however, it is possible to set a range of operational parameters that can be used to maximize extract potential.

Isolation and purification of Taraxacum compounds needs to be further explored. Although synergy is an important characteristic of plant mixtures responsible for its antimicrobial activity and even though bioactive synthesis is difficult and expensive on a large scale, knowing the nature of Taraxacum extracts and the associated antimicrobial mechanisms may provide important advantages in synthesizing specific structures with improved antimicrobial properties.

Contradictory information is available in the data analyzed; however, these discrepancies are probably the result of different procedures, particular considerations, or inaccurate process descriptions. These differences make it quite possible that the results are not directly related to the full antimicrobial potential of Taraxacum but to a limited scope. Therefore, extracts and bioassays must be conducted under a standardized protocol to provide reproducible studies and reliable data comparisons between published articles, which would empower research conducted by authors worldwide and allow for the interrelated study of this genus. In addition, the efficacy of reported biological activity in vitro could be validated with in vivo assays.

Standardization of the entire procedure (sample manipulation, extraction, and further bioassay) is necessary for comparisons of published data and establishing the exact potential of Taraxacum, or any other plant extract, as a commercial antimicrobial agent. The uniformity of an extract is highly susceptible to external factors that influence plant metabolism. This problem could be solved by performing plant breeding techniques with selected Taraxacum species grown under controlled environmental conditions.

Acknowledgments

This work has been supported by Innova Chile CORFO Code FCR-CSB 09CEII-6991 and a doctoral fellowship awarded by the Pontifical Catholic University of Valparaíso, Chile.