Open access peer-reviewed chapter

Taraxacum Genus: Extract Experimental Approaches

By María Eugenia Martínez Valenzuela, Katy Díaz Peralta, Lorena Jorquera Martínez and Rolando Chamy Maggi

Reviewed: November 30th 2017Published: November 5th 2018

DOI: 10.5772/intechopen.72849

Downloaded: 782

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.

Keywords

  • extract
  • antimicrobial activity
  • Taraxacum genus
  • phytochemical bioassay

1. Introduction

Taraxacumhas 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 Taraxacumgenus

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 situantifungal 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 Taraxacumextracts, 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. officinaleshowed no activity in the disc diffusion test but moderate toxicity against E. coliand B. subtilisin the broth dilution test [4]. Considering this issue, a list of the bioassays used for testing Taraxacumextracts against every strain identified, including the main results, is presented in Table 1.

StrainsBioassayResults expresionPositive controlActive concentrationMain resultsReference
Alternaria alternataBroth dilution assayIC50, MIC, Morphological changesNone15 μM2.9 μM; 1.0 μM; +[5]
A. alternataPaper disc diffusion method% InhibitionNoneS, S/2, S/10, S/10016.7–76.2%[6]
A. carbonarius(Bainier) ThomMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X4%, 0%[7]
A. flavus0064Agar tube dilutionInhibition growthTerbinafine 12 mg/mL 100%15 mg/mL70.80%[8]
A. fumigatus66Agar tube dilutionInhibition growthTerbinafine 12 mg/mL 100%16 mg/mL84.80%[8]
A. hidrophila(food poisoning patients)Agar diffusion methodInhibition zoneCephalotin 30 μg/mL (20 mm)10 mg/mLNo activity[9]
A. nigerBroth dilution assayIC50, MIC, Morphological changesNone15 μM4.2 μM; 2.8 μM; +[5]
A. niger0198Agar tube dilutionInhibition growthTerbinafine 12 mg/mL 100%17 mg/mL37.40%[8]
A. nigerUPCC 3701Agar well diffusionInhibition zone, antimicotic indexCanesten (23 mm, 1.3)30 μgNo activity[9]
A. nigervan ThiegemMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X3%, 45%[7]
A. nigerVKM F-33Broth dilution assayIC50 (50% growth inhibition)None6–10 μM1.2–5.6 μM[10]
A. nigerVKM F-33Microtiterd methodIC50None15.6–250 μg/mLNo activity[11]
A. flavusQC 6658Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
A. fumigatusDisk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
A. nigerDisk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
B. cereusAgar diffusion methodInhibition zoneCephalotin 30 μg/mL (22 mm)10 mg/mL18 mm[9]
B. cereus(spoiled rice)Agar diffusion methodInhibition zoneCephalotin 30 μg/mL (20 mm)10 mg/mL18 mm[9]
B. cereusATCC 1778Broth dilution assayMICCloramphenicol (0.004 μM)No information2.5 μM[13]
B. cereusNCTC 7464Broth dilution assayMICNone2 mg/mL500 μg/mL[14]
B. cereusNCTC 7464Microtiterd methodMICNone2 mg/mL250 μg/mL[15]
B. cinéreaMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X9%, 38%[7]
B. cinereaSGR-1Broth dilution assayIC50 (50% growth inhibition)None6–10 μM5.2–5.8 μM[5]
B. cinereaSGR-1Microtiterd methodIC51None15.6–250 μg/mLNo activity[11]
B. sorokinianaBroth dilution assayIC50, MIC, Morphological changesNone15 μM>15 μM; >15 μM;[10]
B. sorokiniana6/10Broth dilution assayIC50 (50% growth inhibition)None6–10 μM5.2 μM[5]
B. sorokinianaF-1446Microtiterd methodIC52None15.6–250 μg/mLNo activity[10, 11]
B. subtilisAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discweak activity, not indicated[16]
B. subtilisAgar inoculationMICNoneNo information7.0 mg/mL[17]
B. subtilisDisc diffusion methodInhibition zoneNoneNo information12.04 mm[18]
B. subtilisDisc diffusion methodInhibition zoneTetracyclin 10 μg/disc (23.6 mm)10 mg/mL5.1–97.9%[19]
B. subtilisBroth dilutionMICTetracyclin (MIC 5.0 μg/mL)10 μg/mL5.1–97.9%[19]
B. subtilisAgar well diffusionInhibition zoneNone120 μg/mL10.0–14.0 mm[20]
B. subtilisBroth dilution assayMICTetracyclin 100 μg/mL (84%)50 mg/mL10–54%[4]
B. subtilisDisc diffusion methodInhibition zoneTetracyclin 100 μg/mL (22 mm)50 mg/mL11–19 mm[4]
B. subtilisATCC 1149Agar well diffusionInhibition zone, antimicotic indexChloramphenicol (8 mm, 0.3)30 μg11 mm, 0.1[9]
B. subtilisATCC 6633Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM18 mg/mLNo activity[8]
B. subtilisATCC 6633Disc diffusion methodInhibition zoneControl (8 mm)1000–2000 μg/mL0–12.5 mm[21]
B. subtilisATCC 6633Broth inhibition method% InhibitionNone1000–2000 μg/mL5.1–97.9%[21]
B. subtilisATCC 6633Broth dilution assayMICNoneNo informationNo activity[22]
B. subtilisKCTC 1021Disc diffusion methodInhibition zoneControl (8 mm)500–2000 μg/mL8.5–12.5 mm[23]
B. subtilisKCTC 1021Broth dilution% InhibitionNone1000–2000 μg/mL5.1–97.9%[23]
B. subtilisVKM 1053Agar diffusion methodInhibition zone6–10 μM0.8–1.2 μM[5]
B. cereusNCTC 7464Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
B. pumilus(wildtype hand isolate)Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
B. subtilisNCTC 10400 (NCIMB 8054)Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
C. albicansDisc diffusion methodInhibition zoneNo informationNo informationNo activity[24]
C. albicansBroth dilution assayMICTetracyclin 100 μg/mL (68%)50 mg/mL0–70%[4]
C. albicansDisc diffusion methodInhibition zoneTetracyclin 100 μg/mL (20 mm)50 mg/mL14–20 mm[4]
C. albicansATCC 10231Paper disc diffusion methodInhibition zoneChloramphenicol 30 mcg (27 mm)50 μL/discNo activity[25]
C. albicansATCC 10231Agar diffusion methodInhibition zoneAnfotericin (0.2 mm)200 μg/mL>200 μg/mL[26]
C. albicansATCC 18804Broth dilution assayMICAnfotericin B (0.0004 μM)No information0.039 μM[13]
C. albicansATCC 90028Agar well diffusionAnfotericin B (100 μg/disc)40 μg3.0 mm[27]
C. albicansUPCC 2168Agar well diffusionInhibition zone, antimicotic indexCanesten (18 mm, 0.3)30 μg12 mm, 0.2[9]
C. glabrataATCC 2001Agar diffusion methodInhibition zoneAnfotericin (0.4 mm)200 μg/mL>200 μg/mL[26]
C. gloesporoidesBroth dilution assayIC50, MIC, Morphological changes15 μM>15 μM; >15 μM;-[10]
C. jejuniBroth dilution assayAdhesion, cytotoxicity, Antibacterial3-sialyllactose (IC50 1.4 mg/mL)500 mg/mLIC50 < 3 mg/mL, <10%, no activity[28]
C. jejuniNCTC 11168 (ATCC 700819)Broth dilution assayMIC, IC50, % InhibitionAmpicillin (IC50 1.61 μg/mL)15 μMNo activity[29]
C. lagenariumDirect inoculationRates of InhibitionControl untreated leavesNo information1.90[30]
C. lagenariumDirect inoculationRates of InhibitionControl untreated leavesNo information12.80[30]
C. neoformansATCC 32608Broth dilution assayMICAnfotericin B (0.0008 μM)No information0.039 μM[13]
C. parapsilepsisATCC 22019Agar diffusion methodInhibition zoneAnfotericin (0.4 mm)200 μg/mL>200 μg/mL[26]
C. sativus(S. Ito and Kurib.)Paper disc diffusion methodInhibition zoneMancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)5 mg/discweak activity, not indicated[31]
C. tropicalisATCC 750Agar diffusion methodInhibition zoneAnfotericin (0.4 mm)200 μg/mL2.0 mm[26]
C. utilisATCC 22023Agar diffusion methodInhibition zoneAnfotericin (0.4 mm)200 μg/mL>200 μg/mL[26]
C. violaceumATCC 12472Quorum sensingInhibition zoneNoneNo information7 mm[32]
C. violaceumATCC 31532Quorum sensingInhibition zoneNoneNo informationNo activity[33]
C. violaceumNTCT 13274Quorum sensingInhibition zoneNoneNo informationNo activity[33]
C. albicansDisk diffusion methodInhibition zoneCiprofloxacin (5 μg/disc)130–200 mg/mL>200 mg/mL[12]
C. glabrataATCC 2001Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
C. kruseiATCC 6258Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
C. parapsilosisATCC 22019Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
C. michiganensesubesp. MichiganenseAc-1144Agar diffusion methodInhibition zone6–10 μM0.8–1.4[5]
Cupriavidussp.Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
E. coccusATCC 13048Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM19 mg/mLNo activity[8]
E. coliDisc diffusion, broth dilutionInhibition zone, MICErythromicin (MIC 27 μg/mL)10–500 μg/mL13.3 mm, MIC 50 μg/mL[34]
E. coliAgar inoculationMICNoneNo information1.0 mg/mL[17]
E. coliAgar diffusion methodInhibition zoneNone0.1–1.0 mg/mL>0.5 mg/mL (1–4 mm)[35]
E. coliAgar diffusion methodInhibition zoneCloramphenicol 10 mg/mL (30.5 mm)50–200 mg/mL5.25–23.5 mm[36]
E. coliDietCFU countControlNo informationInhibition[8]
E. coliDisc diffusion methodInhibition zoneNone1 g/mL10.2–18.5 mm[37]
E. coliDisc diffusion methodInhibition zoneNoneNo information13.21 mm[18]
E. coliDisc diffusion methodInhibition zoneGentamycin 10 μg/disc (18.9 mm)10 mg/mL12.05–14.21 mm[19]
E. coliBroth dilutionMICGentamycin (MIC 1.25 μg/mL)10 μg/mL250–500 μg/mL[19]
E. coliDisc diffusion methodInhibition zoneNo informationNo information11–13 mm[24]
E. coliAgar well diffusionInhibition zoneNone120 μg/mL2.0–3.0 mm[20]
E. coliBroth dilution assayMICTetracyclin 100 μg/mL (78%)50 mg/mL14–62%[4]
E. coliDisc diffusion methodInhibition zoneTetracyclin 100 μg/mL (18 mm)50 mg/mL12–15 mm[4]
E. coli7075Agar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
E. coli8739Agar diffusion methodInhibition zoneNone1 mg/mL>1 mg/mL[38]
E. coliATCC 11229Disc diffusion methodInhibition zoneControl (8 mm)500–2000 μg/mL11–13.5 mm[21]
E. coliATCC 11229Broth inhibition method% InhibitionNone500–2000 μg/mL98.1–100%[21]
E. coliATCC 1229Broth dilution assayMICNoneNo informationNo activity[22]
E. coliATCC 15224Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM20 mg/mLNo activity[8]
E. coliATCC 25322Agar well diffusionInhibition zoneGentamycin240 mg/mL6.5 mm[39]
E. coliATCC 8677Paper disc diffusion methodInhibition zoneTicarcillin 75 mcg (27 mm)50 μL/discNo activity[26]
E. coliATCC 8739Broth dilution assayMICNoneS, S/2Inhibition[40]
E. coliDSM 1103Broth dilution assayMICNone2 mg/mLNo activity[14]
E. coliDSM 1103Microtiterd methodMICNone2 mg/mLNo activity[15]
E. coliKCTC 2441Disc diffusion methodInhibition zoneControl (8 mm)500–2000 μg/mL9.0–12 mm[23]
E. coliKCTC 2441Broth dilution% InhibitionNone1500–2000 μg/mL13–98.4%[23]
E. coliNCTC 25922Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
E. coliNCTC 9001Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
E. coliUPCC 1195Agar well diffusionInhibition zone, antimicotic indexChloramphenicol (25 mm, 3.2)30 μg11 mm, 0.1[9]
E. faecalisIrrigation in situNone7 mg/mLweak activity, not indicated[41]
E. faecalisATCC 19433Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM. Cefixime 1.0 μM21 mg/mLNo activity[8]
E. coli0157 NCTC 12900Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
E. coliDH5Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Enterobacter/Klebsiellasp.Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
E. faecalisNCTC 775Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Exophiala(Wangiella) dermatitidisQC 7895Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
F. avenaceumBroth dilution assayIC50, MIC, Morphological changes15 μM13.1 μM; 6.7 μM; +[10]
F. graminearumVKM F-1668Broth dilution assayIC50 (50% growth inhibition)6–10 μM>10 μM[5]
F. oxysporiumSchlechtPaper disc diffusion methodInhibition zoneMancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)6 μM5.7 μM[31]
F. oxysporiumTSKHA-4Broth dilution assayIC50 (50% growth inhibiton)Kanamycin6 μM5.7 μM[5]
F. oxysporiumTSKHA-4Microtiterd methodIC53None15.6–250 μg/mLNo activity[11]
H. pyloriPaper disc diffusion methodInhibition zoneControl (8 mm)No information10 mm[42]
H. pylori NCTC 11639 (ATCC 43629)Broth dilution assayMIC, IC50, % InhibitionGentamicin (IC50 0.081 μg/mL)500 mg/mL25%[29]
K. pneumoniaeAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
K. pneumoniaeAgar diffusion methodInhibition zoneCloramphenicol 10 mg/mL (26.5 mm)50–200 mg/mL[36]
K. pneumoniaeDisc diffusion methodInhibition zoneGentamycin 10 μg/disc (18.9 mm)10 mg/mL13.24–17.72 mm[19]
K. pneumoniaeBroth dilutionMICGentamycin (MIC 5.0 μg/mL)10 μg/mL125–250 μg/mL[19]
K. pneumoniaeATCC 13866Broth dilution assayMICCloramphenicol (0.001 μM)No information0.625 μM[13]
K. pneumoniaeUC 5Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM. Cefixime 1.0 μM22 mg/mLNo activity[8]
K. aerogenesNCTC 9528Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
K. pneumoniae700,603Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
L. monocytogenesKCCM 40307Disc diffusion method. Broth Inhibition methodInhibition zone. % InhibitionControl (8 mm). None500–2000 μg/mL0–12 mm. 5.1–97.9%[21]
L. monocytogenesKCCM 40307Disc diffusion method. Broth Inhibition methodInhibition zone. % InhibitionControl (8 mm). None500–2000 μg/mL10.5–12 mm. 27.2–94%[23]
L. monocytogenesNCTC 11994Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
M. aureum4721 EBroth dilutionMICStreptomycin (IC50 1.14 μg/mL)500 μg/mL>500 μg/mL[41]
M. bovisBCGBroth dilutionMICStreptomycin (IC50 1.14 μg/mL)500 μg/mL>500 μg/mL[43]
M. canisAgar well diffusion, agar tube dilutionNo informationNo informationNo informationInhibition[44]
M. kristinaeAgar inoculationMICNoneNo information5.0–7.0 mg/mL[17]
M. laxaMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X11%, 5%[7]
M. luteusAgar well diffusionInhibition zoneNone120 μg/mL5.0–9.0 mm[20]
M. luteusATCC 10240Agar diffusion methodMICErythromicin 1.0 μM23 mg/mL1.0 μM[8]
M. piriformisPaper disc diffusion method% InhibitionNoneS, S/2, S/10, S/1005.3–66.7%[6]
M. smegmatisMC2 155Broth dilutionMICStreptomycin (IC50 1.14 μg/mL)120 μg/mL5.3–66.7%[43]
M. tuberulosisH37RABroth dilution methodMICRifampim. Isoniazid. KanamycinNo information> 200 μg/mL[44]
MRSA (clinical isolated)Broth dilution assay. Microtiterd methodMICNone2 mg/mL375 μg/mL. 500 μg/mL[14]
MRSA AARM 3696Disc diffusion methodInhibition zoneGentamicin 0.2 mg/disc (20.1 mm)0.5–3.0 mg/disc6.8–16.5 mm[45]
P. acnesBroth dilution assayNo informationNo informationNo informationNo information[46]
P. aeruginosaBroth dilution assayNo informationNo informationNo informationNo information[46]
P. aeruginosaDisc diffusion, broth dilutionInhibition zone, MICErythromicin10–500 μg/mLNo activity[34]
P. aeruginosaAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
P. aeruginosaAgar diffusion methodInhibition zoneCloramphenicol 10 mg/mL (26.0 mm)50–200 mg/mL[36]
P. aeruginosaDisc diffusion methodInhibition zoneGentamycin 10 μg/disc (20.0 mm)10 mg/mL16.52–19.19 mm[19]
P. aeruginosaBroth dilutionMICGentamycin (MIC 2.5 μg/mL)10 μg/mL125–250 μg/mL[19]
P. aeruginosaAgar well diffusionInhibition zoneNone120 μg/mL8.0–13.0 mm[20]
P. aeruginosaATCC 15442Broth dilution assayMICCloramphenicol (0.015 μM)No information2.5 μM[13]
P. aeruginosaATCC 7221Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM. Cefixime 1.0 μM24 mg/mLNo activity[8]
P. aeruginosaATCC 9027Broth dilution assayMICNoneNo informationNo activity[22]
P. aeruginosaATCC 9721Paper disc diffusion methodInhibition zoneTicarcillin 75 mcg (20 mm)50 μL/discNo activity[25]
P. aeruginosaUPCC 1244Agar well diffusionInhibition zone, antimicotic indexChloramphenicol (23 mm, 2.8)30 μg11 mm, 0.1[9]
P. betaeBroth dilution assayIC50, MIC, Morphological changes15 μM10.7 μM; 8.0 μM; +[10]
P. betaeF-2532Microtiterd methodIC54None15.6–250 μg/mLNo activity[11]
P. debaryanumVKM F-1505Broth dilution assayIC50 (50% growth inhibition)None6–10 μM2.6 μM[5]
P. digitatum(Perss) SaccMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X12%, 42%[7]
P. expansumPaper disc diffusion method% InhibitionNoneS, S/2, S/10, S/10015.7–69.7%[6]
P. expansumLink.Microassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75XNo activity[7]
P. expansumLink. ATCC 42710Paper disc diffusion methodInhibition zoneControl (8 mm)0.1 g/mL12 mm[47]
P. infestansMicrotiterd methodIC55None15.6–250 μg/mLNo activity[11]
P. infestansOSV 12Direct inoculation (potato disc)Disease developmentNone1.3–5.3 μM1.3–5.2 μM[5]
P. italicumWehmerMicroassay method on slidesICG, IGTEOnly in vivo assays (Imazalil, Fenhexamid)0.75X2%, 56%[7]
P. mirabilisAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
P. mirabilisAgar diffusion methodInhibition zoneNone0.1–1.0 mg/mL>0.5 mg/mL (4–10 mm)[35]
P. ovaleBroth dilution assayNo informationNo informationNo informationNo information[46]
P. syringeaeVKM B-1546Agar diffusion methodInhibition zone6–10 μM1.2–1.3 μM[5]
P. vulgarisDisc diffusion, broth dilutionInhibition zone, MICErythromicin (MIC 26 μg/mL)10–500 μg/mL10.1 mm, MIC 100 μg/mL[34]
P. vulgarisAgar inoculationMICNoneNo information5.0 mg/mL[17]
P. vulgarisDisc diffusion methodInhibition zoneGentamycin 10 μg/disc (19.5 mm)10 mg/mL13.38–18.33 mm[19]
P. vulgarisBroth dilutionMICGentamycin (MIC 2.5 μg/mL)10 μg/mL250–500 μg/mL[19]
Penicilliumsp. QC 743275Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Pseudomona sp.Disc diffusion methodInhibition zoneNo informationNo information11–21 mm[24]
P. aeruginosaNCTC 1662Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
P. aeruginosaNCTC 27853Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Pseudomonassp.Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
R. solani18,619Agar tube dilutionInhibition growthTerbinafine 12 mg/mL 100%25 mg/mL77.47%[8]
R. solaniKühnPaper disc diffusion methodInhibition zoneMancozeb, Thiram, Carboxin, Benomyl (1 mg/disc)5 mg/disc4.75–17.63 mm[31]
S. aureusNCTC 8178Microtiterd methodMICNone2 mg/mL500 μg/mL[15]
S. abony entericaNCTC 6017Broth dilution assayMICNoneS, S/2Inhibition[40]
S. agalactiaeDisc diffusion methodInhibition zoneNone1 g/mL11.1–19.7 mm[37]
S. aureusBroth dilution assayNo informationNo informationNo informationNo information[46]
S. aureusDisc diffusion, broth dilutionInhibition zone, MICErythromicin (MIC 33 μg/mL)10–500 μg/mL12.7 mm, MIC 50 μg/mL[34]
S. aureusAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
S. aureusAgar inoculationMICNoneNo information5.0 mg/mL[17]
S. aureusAgar diffusion methodInhibition zoneNone0.1–1.0 mg/mL>0.5 mg/mL (1–4 mm)[35]
S. aureusAgar diffusion methodInhibition zoneCloramphenicol 10 mg/mL (35.0 mm)100–200 mg/mL9.0–10.75 mm[36]
S. aureusDisc diffusion methodInhibition zoneNone1 g/mL9.7–19.9 mm[37]
S. aureusDisc diffusion methodInhibition zoneNoneNo information16.15 mm[18]
S. aureusDisc diffusion methodInhibition zoneTetracyclin 10 μg/disc (38.8 mm)10 mg/mL11.22–15.07 mm[19]
S. aureusBroth dilutionMICTetracyclin (MIC 2.5 μg/mL)10 μg/mL250 μg/mL[19]
S. aureusDisc diffusion methodInhibition zoneNo informationNo information11–21 mm[24]
S. aureusAgar well diffusion, agar tube dilutionNo informationNo informationNo informationInhibition[44]
S. aureusAgar well diffusionInhibition zoneNone120 μg/mL4.0–11.0 mm[20]
S. aureusBroth dilution assayMICTetracyclin 100 μg/mL (75%)50 mg/mL0–76%[4]
S. aureusDisc diffusion methodInhibition zoneTetracyclin 100 μg/mL (17 mm)50 mg/mL9–18 mm[4]
S. aureus(salted white cheese)Agar diffusion methodInhibition zoneCephalotin 30 μg/mL (24 mm)10 mg/mL16 mm[9]
S. aureusATCC 12600Paper disc diffusion methodInhibition zoneChloramphenicol 30 mcg (27 mm)50 μl/discNo activity[25]
S. aureusATCC 25922Agar well diffusionInhibition zonePenicillin320 mg/mL10.4 mm[39]
S. aureusATCC 43300Disc diffusion methodInhibition zoneErythromycin 50 μg/well40 μg7.5 mm[27]
S. aureusATCC 6530Broth dilution assayMICNoneNo informationNo activity[22]
S. aureusATCC 6538Broth dilution assayMICCloramphenicol (0.063 μM)No information5.0 μM[13]
S. aureusATCC 6538Broth dilution assayMICNoneS, S/2No activity[40]
S. aureusATCC 6538Agar diffusion methodInhibition zone, MICErythromicin 1.0 μM. Cefixime 1.0 μM26 mg/mLNo activity[8]
S. aureusATCC 6538Disc diffusion methodInhibition zoneControl (8 mm)1500–2000 μg/mL0–12 mm[22]
S. aureusATCC 6538Broth inhibition method% InhibitionNone1500–2000 μg/mL5.1–97.9%[22]
S. aureusATCC 6538Agar diffusion methodInhibition zoneNone1 mg/mL>1 mg/mL[38]
S. aureusKCTC 1916Disc diffussion methodInhibition zoneControl (8 mm)500–2000 μg/mL8.5–13.5 mm[24]
S. aureusKCTC 1916Broth dilution% InhibitionNone500–2000 μg/mL98.1–100%[24]
S. aureusKCTC 3881Disc diffussion methodInhibition zoneGentamicin 0.2 mg/disc (20.1 mm)0.5–3.0 mg/disc6.8–16.5 mm[45]
S. aureusNCTC 8178Broth dilution assayMICNone2 mg/mL375 μg/mL[14]
S. aureusUPCC 1143Agar well diffussionInhibition zone, antimicotic indexChloramphenicol (20 mm, 2.3)30 μgNo activity[9]
S. australisBroth dilution assayGrowthNone10–10,000 ppmNo activity[46]
S. cerviseaeBroth dilution assayMICTetracyclin 100 μg/mL (50%)50 mg/mL0–64%[4]
S. cerviseaeDisc diffussion methodInhibition zoneTetracyclin 100 μg/mL 18 mm)50 mg/mL12–15 mm[4]
S. dysgalactiaeDisc diffusion methodInhibition zoneNone1 g/mL13.8–9.6 mm[37]
S. enterica sorovar typhimuriumATCC 13311Broth dilution assayMICCloramphenicol (0.001 μM)No information5.0 μM[13]
S. enteriditisDisc diffussion methodInhibition zoneNo informationNo informationNo activity[24]
S. epidermisKCTC 1917Disc diffussion methodInhibition zoneGentamicin 0.2 mg/disc (24.4 mm)130–200 mg/mL7.3–16.7 mm[45]
S. fiexneriDisc diffusion, broth dilutionInhibition zone, MICErythromicin10–500 μg/mLNo activity[34]
S. haemolyticusAgar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discWeak activity, not indicated[16]
S. marscensAgar inoculationMICNoneNo information1.0–5.0 mg/mL[17]
S. sonneiATCC 11060Broth dilution assayMICCloramphenicol (0.001 μM)No information2.5 μM[13]
S. tiphimuriumSARB 69Broth dilution assayMICNone2 mg/mLNo activity[14]
S. typhiAgar well diffussion, agar tube dilutionNo informationNo informationNo informationInhibition[44]
S. typhi(food poisoning patients)Agar diffusion methodInhibition zoneCephalotin 30 μg/mL (18 mm)10 mg/mL14 mm[9]
S. typhiH.Agar diffusion methodInhibition zoneGentamicyn 1 mg/disc, Tetracyclin 2 mg/disc4–12 μg/discNo activity[16]
S. typhimuriumReference collection B-69Microtiterd methodMICNone2 mg/mLNo activity[15]
Salmonella poonaNCTC 4840Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Scedosporium apiospermumQC 7870Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Serratia marcescensDisk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Serratia/Rahnellasp.Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
StaphylococcusDietCFU countControlNo informationInhibition[8]
S. aureus(MRSA) 43,300Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
S. aureus(MSSA) 25,923Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
S. aureusNCTC 6571Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
Staphylococcus epidermidisNCTC 14990Disk diffusion methodInhibition zoneCiprofloxacin 5 μg/disc130–200 mg/mL>200 mg/mL[12]
T. longifususAgar well diffusion, agar tube dilutionNo informationNo informationNo informationInhibition[45]
T. mentagrophytesUPCC 4193Agar well diffusionInhibition zone, antimicotic indexCanesten (55 mm, 4.3)30 μg12 mm, 0.2[9]
V. albo-atrumBroth dilution assayIC50, MIC, Morphological changesNone15 μM>15 μM; >15 μM; −[10]
V. albo-atrum F-2437Microtiterd methodIC56None15.6–250 μg/mLNo activity[11]
V. choleraATCC 11623Agar diffusion methodMICErythromicin 1.0 μM. Cefixime 1.0 μM27 mg/mL12.5 μM[8]
V. parahaemolyticusKCTC 2471Disc diffusion method. Broth Inhibition methodInhibition zone. % InhibitionControl (8 mm). None.500–2000 μg/mL9.5–15 mm.
5.1–97.9%
[21]
V. parahaemolyticusKCTC 2471Disc diffusion method. Broth dilution.Inhibition zone. % InhibitionControl (8 mm). None.500–2000 μg/mL9.5 - 15 mm.
84.0–97%
[23]
X. campestrisVKM −608Broth dilution assayIC50 (50% growth inhibition)6–10 μM1.0–1.2 μM[5]

Table 1.

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

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2. Taraxacumextracts versus commercial antibiotics

When comparing Taraxacumextracts to commercial antibiotics, C. jejuniadhesion was controlled by a Taraxacumextract 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. officinaleextract 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. luteuswas 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 Rhizoctoniaspp. 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. mongolicumflower, 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. officinaleextracts 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 Taraxacumextract needed a concentration of 5 mg/disc to achieve the same effect [31]. For H. pyloriand 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. colias 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. coliand S. aureuswere lower compared to an ethanolic extract of T. officinaleat 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. officinaleat 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 Taraxacumextracts exhibited no activity under the tested conditions. For instance, embedded discs with 50 μL of an ethanolic extract of T. officinalewere 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. officinaleleaves and roots (chloroform, methanol, and water) were not active towards Mycobacteriumcompared to streptomycin at 1.14 μg/mL [43]. An ethanolic extract of T. phaleratumwas 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. officinaleat 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 Taraxacumextracts that achieve inhibitions similar to the synthetic antibiotics are 100–10,000 times higher, which makes Taraxacumextracts 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 Taraxacumgenus 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 Taraxacumwith 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 Taraxacumextracts can be performed reliably for testing antimicrobial activity, the real potential of the Taraxacumgenus 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].

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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 vivosituation. 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 Taraxacumgenus 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 Taraxacumbeen 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 Taraxacumindicates 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. aureususing 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.

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4. Scaling up from in vitro to in vivo assays

Scaling up an antimicrobial assay from controlled, in vitroconditions to that of natural, in vivoconditions can be difficult if no proper considerations are taken. For instance, active concentrations for in vitroconditions frequently cannot be reached in vivobecause the infecting microorganisms are never exposed to constant concentrations of an antimicrobial agent. Microorganisms in vivoare 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 vivosystem. 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 vivoresponses to Taraxacumextracts (Chapter 1; see Section 2.2.2), but studies in animal tissues and organs have not yet been performed directly.

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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 Taraxacumgenus 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 Taraxacumstudies 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, Taraxacumspp., 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 Taraxacumextracts; 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 Taraxacumvary 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 Taraxacumplants have been used in antimicrobial research. Only one Taraxacumstudy indicated differences between a root extract and a leaf extract, in which the root extract was active against S. aureusand S. typhi.Extracts of plant roots and herbs of different Taraxacumspecies endemic to Turkey displayed significant activity against M. canisand T. longifusus[44]. Few studies refer to the antimicrobial properties of Taraxacumderivatives. Pseudomonasp., S. aureus, and E. coliwere inhibited in a disc diffusion assay, but C. albicansand S. enteriditiswere not inhibited by T. officinalehoney [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), Taraxacumroot 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 Taraxacumbecause 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 Taraxacumranged 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 Taraxacumplant 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 Taraxacumextracts 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 Taraxacumantimicrobial activity is presented in Tables 3 and 4.

Taraxacumparts mentioned in the textNumber of extracts testedPositive antimicrobial activityNegative
antimicrobial activity
Root511711%3443%
Leaves382818%1013%
Flower13107%34%
Honey643%23%
Herb332%00%
Aerial322416%810%
Whole plant800%810%
No information816643%1519%
Total23215280

Table 2.

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

Polarity index9.08.27.77.46.36.36.26.05.95.65.55.45.15.14.44.14.03.10.0
Genus/solventWaterEthanol 20%Ethanol 35%Ethanol 40–- 45%Ethanol 70– 75%Methanol 70%Acetic acidEthanol 80%Methanol 80%Ethanol 90%Methanol 90%Ethanol 96–100%Methanol 100%AcetoneEthylacetateChloroformDietylether 80%DichloromethaneHexane
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 Taraxacumgenus.

(+) 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 testedPositive antimicrobial activityNegative antimicrobial activity
Low polarity (0–3.0)47229%2510%
Medium polarity (3.1–6.0)1007028%3012%
High polarity (6.1–9.0)1013815%6325%
Total248130118

Table 4.

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

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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 Taraxacumcompounds 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 Taraxacumparts and antimicrobial activities.

Nevertheless, Taraxacumhas been proven effective against most known strains of bacteria, fungi, and protozoa that attack animals and plants through an in vitroor in vivoapproach. All studies of Taraxacumextracts 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. lagenariumfor cucumber or S. australisfor salmonids) were also tested in vivo. For humans, only antimicrobial in vitroassays 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 vivoevidence 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 Taraxacumbiochemical 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 Taraxacumantibiotics. The next step is to test these extracts on in vitroand in vivosystems to establish pharmacodynamics and interactions, facilitating the commercial attractiveness of Taraxacumto the pharmaceutical industry.

The bioavailability, pharmacodynamics, and action mechanisms in Taraxacumbioactives have not yet been addressed. Considering that primarily in vitroand, to a much lesser extent, in vivostudies have been conducted using Taraxacumextracts, 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 Taraxacumextract use in pharmacological approaches.

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7. Conclusion

Only a minor fraction of the Taraxacumspecies 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 Taraxacumcompounds 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 Taraxacumextracts 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 Taraxacumbut 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 vitrocould be validated with in vivoassays.

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 Taraxacumspecies grown under controlled environmental conditions.

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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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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María Eugenia Martínez Valenzuela, Katy Díaz Peralta, Lorena Jorquera Martínez and Rolando Chamy Maggi (November 5th 2018). Taraxacum Genus: Extract Experimental Approaches, Herbal Medicine, Philip F. Builders, IntechOpen, DOI: 10.5772/intechopen.72849. Available from:

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