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 (4C), room temperature (20–25C), and solvent boiling point (50–100C) have been reported for Taraxacum. The majority of the work was conducted in the range of 20–40C, where the maceration process was proposed and, to a lesser extent, extraction under boiling temperatures has also been indicated (80–100C, 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 Polarity index

Genus/solvent

Water

Ethanol20%

Aeromona

Alternaria Aspergillus

Bacillus

Bipolaris

Botrytis Campylobacter

Candida Chromobacterium

Cochilibus Colletotrichum

Cupriavidus

Clavibacter Enterobacter

Enterococcus

Escherichia

Exophiala

Fusarium

Helicobacter

Klebsiella

—

+

—

/+

—

/+

 +

—

+

 +

—

+

—

/+

/+

—

 + /+

293

 —

 +

 +

 +

+

—

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

/+

—

—

+

—

+

 + —

+

—

—

 —

+

 + +

/+

+

—

 +

+

/+

+

 +

 + /+

+

 +

 +

/+

/+

 +

 +

 +

+

/+

—

+

+

 +

Ethanol35%

Methanol70%

Aceticacid

Ethanol80%

Methanol80%

Ethanol90%

Methanol90%

Methanol100%

100% – Ethanol96

Acetone

Ethylacetate

—

—

Chloroform

Dietylether80%

Dichloromethane

Hexane

45% – Ethanol40

75% – Ethanol70

 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 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 Ethanol20% Ethanol35% 45% – Ethanol40 75% – Ethanol70 Methanol70% Aceticacid Ethanol80% Methanol80% Ethanol90% Methanol90% 100% – Ethanol96 Methanol100% Acetone Ethylacetate Chloroform Dietylether80% Dichloromethane Hexane Aeromona — Alternaria + Aspergillus — + + — Bacillus /+ + + + + /+ /+ + + + + /+ Bipolaris /+ Botrytis + + Campylobacter + + Candida — — + /+ + + Chromobacterium — + — Cochilibus + Colletotrichum + — Cupriavidus — Clavibacter + + Enterobacter — — Enterococcus /+ Escherichia /+ + — + + — + — /+ /+ + + + + — Exophiala — Fusarium — /+ — Helicobacter + + Klebsiella — — /+

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].

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 (4C), room temperature (20–25C), and solvent boiling point (50–100C) have been reported for Taraxacum. The majority of the work was conducted in the range of 20–40C, where the maceration process was proposed and, to a lesser extent, extraction under boiling temperatures has also been indicated (80–100C, 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

5.6. Relationship between temperature and extraction time

292 Herbal Medicine


Polarity index

Genus/solvent

Water

Ethanol20%

Serratia

Shigella

Staphylococcus

Trichophyton

Vibrio

Xanthomonas

(+) Positive

Table 3.

Antimicrobial

 activity regarding the solvent tested with

antimicrobial

 activity report. (

) No

antimicrobial

 activity reported. Empty cells indicate no study has been performed so far.

Taraxacum genus.

*Taraxacum* Genus: Extract Experimental Approaches http://dx.doi.org/10.5772/intechopen.72849 295

/+

—

 —

+

 + —

+

/+

—

/+

+

+

/+

 +

 +

 +

/+

 +

—

—

—

Ethanol35%

Methanol70%

Aceticacid

Ethanol80%

Methanol80%

Ethanol90%

Methanol90%

Methanol100%

+

+

100% – Ethanol96

Acetone

Ethylacetate

Chloroform

Dietylether80%

Dichloromethane

Hexane

45% – Ethanol40

75% – Ethanol70

 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


Polarity index

Genus/solvent

Water

Ethanol20%

Listeria

Micrococcus Microsporum

Monilinia Mycobacterium

Mucor Penicillium

Pityrosporum

Phoma Phytophthora

Proteus

Pseudomona

Salmonella

Saprolegnia

Pythium

Rhizoctonia Saccharomyces

Salmonella Scedosporium

—

+

—

+

+

—

 —

—

/+

—

—

+ +

 +

—

/+

/+

—

 + +

+

—

/+

+

+

—

—

+

+

+

 +

/+

/+ +

+

 +

+

—

—

Ethanol35%

Methanol70%

Aceticacid

Ethanol80%

Methanol80%

Ethanol90%

Methanol90%

Methanol100%

+

+

+

 —

+

 +

 +

100% – Ethanol96

Acetone

Ethylacetate

Chloroform

Dietylether80%

Dichloromethane

Hexane

45% – Ethanol40

75% – Ethanol70

 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

294 Herbal Medicine

Table 3. Antimicrobial activity regarding the solvent tested with Taraxacum genus. 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.

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

Table 4. Summary of the antimicrobial results regarding the polarity of the Taraxacum extracts tested in main studies.

Number of extracts tested Positive antimicrobial

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%

activity

Negative antimicrobial activity

297

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

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

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

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

regarding Taraxacum antimicrobial activity is presented in Tables 3 and 4.

Total 248 130 118

temperature) among others, complicate the comparison of published data.

6. Perspectives of potential bioassays

Solvents used in Taraxacum

extracts

and antimicrobial activities.

#### 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


Table 4. Summary of the antimicrobial results regarding the polarity of the Taraxacum extracts tested in main studies.

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.
