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.

#### 5.1. Plant material collection

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

288 Herbal Medicine

here provides a promising case for the advantageous commercial usage of this genus.

4. Scaling up from in vitro to in vivo assays

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.

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 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


Table 2. Summary of the antimicrobial results regarding Taraxacum plant parts tested in main studies.


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

*Taraxacum* Genus: Extract Experimental Approaches http://dx.doi.org/10.5772/intechopen.72849 291

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.

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 freezedried. 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.

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

available mentions proper, expert identification.

5.3. Plant part utilization

5.4. Sample manipulation

5.5. Extraction procedure

Figure 1. Reported extraction conditions to achieve positive antimicrobial results.

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.
