6. Perspectives of potential bioassays

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.

vones, polyphenols, tannins, and saponins [58].

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, fla-

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

5.8. Solvents

296 Herbal Medicine

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.

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

This work has been supported by Innova Chile CORFO Code FCR-CSB 09CEII-6991 and a

, Katy Díaz Peralta<sup>2</sup>

1 Escuela de Ingeniería Bioquímica, Facultad de Ingeniería, Pontificia Universidad Católica de

2 Departamento de Química, Universidad Técnico Federico Santa María, Valparaíso, Chile

[1] Radulović N, Blagojević P, Stojanović-Radić Z, Stojanović N. Antimicrobial plant metabolites: Structural diversity and mechanism of action. Current Medicinal Chemistry.

3 Escuela de Ingeniería en Construcción, Facultad de Ingeniería, Pontificia Universidad

, Lorena Jorquera Martínez<sup>3</sup> and

*Taraxacum* Genus: Extract Experimental Approaches http://dx.doi.org/10.5772/intechopen.72849 299

doctoral fellowship awarded by the Pontifical Catholic University of Valparaíso, Chile.

grown under controlled environmental conditions.

Acknowledgements

Author details

References

Rolando Chamy Maggi<sup>1</sup>

Valparaíso, Valparaíso, Chile

María Eugenia Martínez Valenzuela<sup>1</sup>

Católica de Valparaíso, Valparaíso, Chile

\*

\*Address all correspondence to: rolando.chamy@pucv.cl

2013;20:932-952. DOI: 10.2174/092986713805219136

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.
