The combination of both views, including carboxylate efflux, the accumulation of carboxylates in the rhizosphere soil, the chemistry of P mobilization and the uptake of mobilized P by the roots may help to evaluate the contribution of carboxylate excretion to P acquisition by higher plants.

For this purpose, mathematical models are a useful tool. Also, such models allow, by the aid of sensitivity analysis, to evaluate the contribution of different parameters to the acquisition of P by higher plants.

The Excretion of Di- and Tricarboxylic Acids by Roots of Higher Plants Can Strongly Improve the… http://dx.doi.org/10.5772/intechopen.73033 61

**Table 1.** Maximum phosphate and iron + aluminum mobilization by organic anions.

The combination of both views, including carboxylate efflux, the accumulation of carboxylates in the rhizosphere soil, the chemistry of P mobilization and the uptake of mobilized P by the roots may help to evaluate the contribution of carboxylate excretion to P acquisition by

**Figure 8.** Relation between the citrate concentration in soil and the P solubility at different pH in a humic Podzol.

**Figure 7.** Relation between the citrate concentration in soil and the P solubility at different pH in a Ferralsol. (Modified

For this purpose, mathematical models are a useful tool. Also, such models allow, by the aid of sensitivity analysis, to evaluate the contribution of different parameters to the acquisition

higher plants.

from Gerke [24]).

60 Carboxylic Acid - Key Role in Life Sciences

of P by higher plants.

(Modified from Gerke [24]).

Nye [60, 61] developed a mathematical model to quantitatively describe the influx of P as affected by the efflux of mobilizing agents.

Gerke [24] was the first who used the mathematical framework of Nye [60, 61] to quantify the influx of mobilized P as a result of a definitive carboxylate efflux. Some of the results of the calculations are shown in **Figures 9** and **10**.

For the three plant species such as rye grass, white clover and red clover, the maximum citrate efflux was used in the calculations (from **Figures 2**, **4**, and **6**). For the accumulation of citrate in soil, we assumed two strongly differing values in order to include a possible range of accumulation (see in detail Gerke [24]).

**Figure 9.** Influx of mobilized P as related to maximum citrate efflux of plants grown in a Ferralsol by (a) rye grass, (b) white clover and (c) red clover. (Modified from Gerke [24]).

of P-sorbing surfaces is initiated. In this context, simultaneous acidification and carboxylate excretion may increase the dissolution of P-sorbing sites more efficient than carboxylate

The Excretion of Di- and Tricarboxylic Acids by Roots of Higher Plants Can Strongly Improve the…

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63

Finally, the role of root-derived carboxylates on the acquisition of P esters should be considered. This topic has been reviewed by Gerke [20]. In general, phosphate monoesters may accumulate in soils among which higher phosphorylated inositol phosphates account for the

Worldwide, more than 51 millions of tons of phytate (myoinositol hexakisphosphate) are annually produced in crops, seeds and fruits, which is equivalent to about 66% of the P annually applied to agricultural land [62]. Higher phosphorylated inositol phosphates are strongly bound to the soil solid phase often much stronger than the orthophosphate anion [20, 63]. The

Adams and Pate [64] showed that in soil, white lupin but not narrow leaf lupin can acquire P from phytate. White lupin forms root clusters with extensive citrate excretion, but narrow leaf

The mobilization by carboxylates is the rate limiting step in acquisition of P from phytate and similar molecules and not the reduced activity of phytases. For a detailed discussion, see

The partly contrasting results concerning the role of root-released carboxylates on P acquisition by higher plants are mainly due to deficits in the concepts of research and experimental

Carboxylates will affect the soil P solubility after adsorption to the soil solid phase and not dissolved in the soil solution. It is a misconception to consider exclusively the carboxylate concentration in the rhizosphere soil solution and not the carboxylate concentration in the soil solid phase. The determination of carboxylate bound to the soil solid phase is often ignored or is determined by deficient methods. Often, weak extractants such as dilute acids or dilute salt solutions are used, which extract a very low proportion of rhizosphere soil-bound carboxylates. Considering the range of carboxylate efflux of P-deficient plants, especially cluster root forming plants such as white and yellow lupin, and species such as red clover, alfalfa, sugar beet

Citrate and, to some extent, oxalate are the anions, which are most efficient with respect to P

The most important organic P forms in soil, higher phosphorylated inositol phosphates are strongly bound to the soil solid phase and can be mobilized by carboxylates similar to the

and spinach may strongly mobilize the soil P and acquire the mobilized P.

initial step of P acquisition from inositol phosphates is its mobilization.

excretion alone [21].

dominant proportion of ester-P in many soils [20].

lupin forms no root clusters [27].

Gerke [20].

methods.

mobilization.

orthophosphate anion.

**2. Conclusions**

**Figure 10.** Influx of mobilized P as related to citrate efflux of plants grown in a humic Podzol by (a) rye grass, (b) white clover and (c) red clover. (Modified from Gerke [24]).

In none of the soils at either citrate accumulation level did the maximum citrate efflux of rye grass have any effect on the acquisition of P. The high overall citrate efflux of P-deficient red clover strongly increased the influx of mobilized P over the required level in all cases. White clover with a medium citrate efflux strongly increased the P influx of mobilized P in a soil with humic-Al(Fe)-P complexes as dominant P fraction. Compared to soils with mainly Al(Fe) oxide-P complexes, humic-associated P is much easier to mobilize by carboxylates [46, 58].

The results shown in **Figures 9** and **10** show that citrate excretion by P-deficient roots is an exceptional efficient way to improve P acquisition in P-fixing soils.

In their review on P efficiency, Richardson et al. [44] presented a separate chapter titled: "Can the release of organic anions from roots mobilize phosphorus?"

In this chapter, Richardson et al. [44] related [5–50 μmol citrate/g rhizosphere soil] to 1–10 [mM] citrate in the soil solution and then stated that 1 [mM] citrate or more is effective for P mobilization in some soils. This view on the rhizosphere chemistry of carboxylates and phosphate is misleading for at least two reasons.

First, the buffering of carboxylates in soil is a strongly varying soil parameter and cannot be generalized to a relation of 1–10 [mM] citrate versus 5–50 [μmol citrate/g soil], suggesting this as the general range of citrate buffering in all soils. Gerke [17, 24] showed for a strongly anionfixing Ferralsol, that citrate concentration in the soil solution of less than 10 μM corresponds to more than 60 [μmol citrate/g soil] at the soil solid phase suggesting a much wider range of citrate buffering.

Second, it is a misleading view that carboxylates in the soil solution are responsible for the mobilization of P. The initial or essential step in P mobilization is the adsorption of the carboxylates to the soil solid phase where the carboxylate-mediated P desorption or dissolution of P-sorbing surfaces is initiated. In this context, simultaneous acidification and carboxylate excretion may increase the dissolution of P-sorbing sites more efficient than carboxylate excretion alone [21].

Finally, the role of root-derived carboxylates on the acquisition of P esters should be considered. This topic has been reviewed by Gerke [20]. In general, phosphate monoesters may accumulate in soils among which higher phosphorylated inositol phosphates account for the dominant proportion of ester-P in many soils [20].

Worldwide, more than 51 millions of tons of phytate (myoinositol hexakisphosphate) are annually produced in crops, seeds and fruits, which is equivalent to about 66% of the P annually applied to agricultural land [62]. Higher phosphorylated inositol phosphates are strongly bound to the soil solid phase often much stronger than the orthophosphate anion [20, 63]. The initial step of P acquisition from inositol phosphates is its mobilization.

Adams and Pate [64] showed that in soil, white lupin but not narrow leaf lupin can acquire P from phytate. White lupin forms root clusters with extensive citrate excretion, but narrow leaf lupin forms no root clusters [27].

The mobilization by carboxylates is the rate limiting step in acquisition of P from phytate and similar molecules and not the reduced activity of phytases. For a detailed discussion, see Gerke [20].

## **2. Conclusions**

In none of the soils at either citrate accumulation level did the maximum citrate efflux of rye grass have any effect on the acquisition of P. The high overall citrate efflux of P-deficient red clover strongly increased the influx of mobilized P over the required level in all cases. White clover with a medium citrate efflux strongly increased the P influx of mobilized P in a soil with humic-Al(Fe)-P complexes as dominant P fraction. Compared to soils with mainly Al(Fe) oxide-P complexes, humic-associated P is much easier to mobilize by carboxylates [46, 58].

**Figure 10.** Influx of mobilized P as related to citrate efflux of plants grown in a humic Podzol by (a) rye grass, (b) white

The results shown in **Figures 9** and **10** show that citrate excretion by P-deficient roots is an

In their review on P efficiency, Richardson et al. [44] presented a separate chapter titled: "Can

In this chapter, Richardson et al. [44] related [5–50 μmol citrate/g rhizosphere soil] to 1–10 [mM] citrate in the soil solution and then stated that 1 [mM] citrate or more is effective for P mobilization in some soils. This view on the rhizosphere chemistry of carboxylates and phosphate is

First, the buffering of carboxylates in soil is a strongly varying soil parameter and cannot be generalized to a relation of 1–10 [mM] citrate versus 5–50 [μmol citrate/g soil], suggesting this as the general range of citrate buffering in all soils. Gerke [17, 24] showed for a strongly anionfixing Ferralsol, that citrate concentration in the soil solution of less than 10 μM corresponds to more than 60 [μmol citrate/g soil] at the soil solid phase suggesting a much wider range of

Second, it is a misleading view that carboxylates in the soil solution are responsible for the mobilization of P. The initial or essential step in P mobilization is the adsorption of the carboxylates to the soil solid phase where the carboxylate-mediated P desorption or dissolution

exceptional efficient way to improve P acquisition in P-fixing soils.

the release of organic anions from roots mobilize phosphorus?"

misleading for at least two reasons.

clover and (c) red clover. (Modified from Gerke [24]).

62 Carboxylic Acid - Key Role in Life Sciences

citrate buffering.

The partly contrasting results concerning the role of root-released carboxylates on P acquisition by higher plants are mainly due to deficits in the concepts of research and experimental methods.

Carboxylates will affect the soil P solubility after adsorption to the soil solid phase and not dissolved in the soil solution. It is a misconception to consider exclusively the carboxylate concentration in the rhizosphere soil solution and not the carboxylate concentration in the soil solid phase.

The determination of carboxylate bound to the soil solid phase is often ignored or is determined by deficient methods. Often, weak extractants such as dilute acids or dilute salt solutions are used, which extract a very low proportion of rhizosphere soil-bound carboxylates.

Considering the range of carboxylate efflux of P-deficient plants, especially cluster root forming plants such as white and yellow lupin, and species such as red clover, alfalfa, sugar beet and spinach may strongly mobilize the soil P and acquire the mobilized P.

Citrate and, to some extent, oxalate are the anions, which are most efficient with respect to P mobilization.

The most important organic P forms in soil, higher phosphorylated inositol phosphates are strongly bound to the soil solid phase and can be mobilized by carboxylates similar to the orthophosphate anion.

The quantitative effect of carboxylate excretion on the acquisition of soil P by higher plants requires an integrative concept, including all relevant soil and plant parameters. Such a framework may be a mathematical model based on experimentally determined parameters.

[11] Hinsinger P. Bioavailability of soil inorganic P in the rhizosphere as affected by root-

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## **Author details**

#### Jörg Gerke

Address all correspondence to<EMAIL_ADDRESS>
Reetwiesenhof, Rukieten, Germany

## **References**

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The quantitative effect of carboxylate excretion on the acquisition of soil P by higher plants requires an integrative concept, including all relevant soil and plant parameters. Such a framework may be a mathematical model based on experimentally determined parameters.

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Address all correspondence to<EMAIL_ADDRESS>
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Reetwiesenhof, Rukieten, Germany

Jörg Gerke

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**Provisional chapter**

**Isolation and GC Analysis of Fatty Acids: Study Case of**

**Isolation and GC Analysis of Fatty Acids: Study Case of**

*Urtica dioica* L. is perennial plant with remarkable medical properties and has been widely used in folk medicine and as a food. Fatty acids presented in its leaves were extracted applying three different techniques: classic, automated Soxhlet, and supercritical fluid extraction (SFE). SFE was performed at three different pressures (100, 200, and 300 bar) and two different temperatures (40 and 60°C). Obtained extract was analyzed using GC-FID analytical technique in order to obtain fatty acid profile samples. The highest yield was obtained in the case of automated Soxhlet extraction (218.907 mg/g), while the lowest was observed in the case of classic extraction (15.031 mg/g). Soxhlet approach provided the highest yield of saturated fatty acids (173.348 mg/g), while supercritical fluid extraction gave better results in the case of unsaturated fatty acids (60.062 mg/g). Deeper analysis of SFE extracts revealed relationship between extraction parameters (temperature and pressure) and yields of fatty acids where lower pressure ensured higher yield of unsaturated while higher pressure gave higher yield of saturated acids. On the other hand, increasing in temperature at isobaric conditions negatively influenced the yield of

fatty acids except in the case of 300 bar where yield was higher at 60°C.

**Keywords:** stinging nettle, leaves, extraction, fatty acids, GC-FID analysis

*Urtica dioica* L. (stinging nettle) is perennial, wild-growing plant from Urticaceae botanical family [1]. This plant is generally known for biological activity and positive effect which expresses on human health. It has long history of application in folk medicine where it has

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.73533

**Stinging Nettle Leaves**

**Stinging Nettle Leaves**

Saša Đurović, Saša Šorgić, Saša Popov, Marija Radojković and Zoran Zeković

Saša Đurović, Saša Šorgić, Saša Popov, Marija Radojković and Zoran Zeković

http://dx.doi.org/10.5772/intechopen.73533

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Isolation and GC Analysis of Fatty Acids: Study Case of Stinging Nettle Leaves Isolation and GC Analysis of Fatty Acids: Study Case of Stinging Nettle Leaves**

DOI: 10.5772/intechopen.73533

Saša Đurović, Saša Šorgić, Saša Popov, Marija Radojković and Zoran Zeković Saša Đurović, Saša Šorgić, Saša Popov, Marija Radojković and Zoran Zeković

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73533

#### **Abstract**

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68 Carboxylic Acid - Key Role in Life Sciences

**34**:677-691

*Urtica dioica* L. is perennial plant with remarkable medical properties and has been widely used in folk medicine and as a food. Fatty acids presented in its leaves were extracted applying three different techniques: classic, automated Soxhlet, and supercritical fluid extraction (SFE). SFE was performed at three different pressures (100, 200, and 300 bar) and two different temperatures (40 and 60°C). Obtained extract was analyzed using GC-FID analytical technique in order to obtain fatty acid profile samples. The highest yield was obtained in the case of automated Soxhlet extraction (218.907 mg/g), while the lowest was observed in the case of classic extraction (15.031 mg/g). Soxhlet approach provided the highest yield of saturated fatty acids (173.348 mg/g), while supercritical fluid extraction gave better results in the case of unsaturated fatty acids (60.062 mg/g). Deeper analysis of SFE extracts revealed relationship between extraction parameters (temperature and pressure) and yields of fatty acids where lower pressure ensured higher yield of unsaturated while higher pressure gave higher yield of saturated acids. On the other hand, increasing in temperature at isobaric conditions negatively influenced the yield of fatty acids except in the case of 300 bar where yield was higher at 60°C.

**Keywords:** stinging nettle, leaves, extraction, fatty acids, GC-FID analysis

## **1. Introduction**

*Urtica dioica* L. (stinging nettle) is perennial, wild-growing plant from Urticaceae botanical family [1]. This plant is generally known for biological activity and positive effect which expresses on human health. It has long history of application in folk medicine where it has

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

been used for treatment of flailing arthritis or paralytic limbs, stimulation of circulation, and warming the joints and extremities [2]. There are wide ranges of studies which have dealt with biological activity of this plant. They have proved antioxidant, antimicrobial, anti-inflammatory, antiulcer, and analgesic properties of plant and its extracts [2–7]. Besides application in medicine, this plant has also been used in human nutrition for a long time. It has been harvested commercially for high content of chlorophylls, which has been used as coloring agent (E140) in food and medical products [8].

relies on application of pressure to maintain water in liquid state during its heating above boiling point. Such state causes decrease in dielectric constant of water together with the polarity. Thus, polarity may, under certain conditions, become close to polarity of methanol [26–28].

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Supercritical fluid extraction is a suitable substitution for conventional approaches such as hydrodistillation, steam distillation, and solvent extraction [29]. Techniques have started its commercial application during the 1980s [30]. Industrial scale of this technique found its application in processes such as decaffeination of green coffee beans and black tea leaves, production of hop extracts, isolation of essential oils, oleoresins and flavoring compounds from natural sources, extraction and fractionation of edible oils, and removal the pesticides from plant materials [31, 32]. This technique relies on application of fluids in their supercritical state. Such state can be achieved at pressures and temperatures above critical ones for given fluid. When this state has been achieved, fluid expressed properties between those characteristic for gas and liquid state [33, 34]. Density of fluid is similar to the density of liquids, while viscosity is similar to the values for gas. Behavior of two important factors influences the diffusivity which ranges between values for liquid and gas state. This results in better transport properties [34]. It should be mentioned that most important characteristic of fluids in this state is the ability to modulate their properties throughout modulation of density of fluid itself. This may be achieved by changing the pressure and temperature of system. Such changes in density influence directly the solubility of desired compounds in the fluid [35]. Carbon dioxide is the most commonly used fluid for this technique due to its nontoxic and nonexplosive properties, low price, availability, easy removable from extracts, and moderate

Due to importance of fatty acids for proper function of human organism, it is also important to establish their occurrence in nature and possibility for utilization. The main goal of this research was to isolate and establish fatty acid profile in stinging nettle leaves. Different extraction techniques were used in order to compare their efficiency for isolation of fatty acids, while GC-FID technique was applied for their quantification. From the presented results, relationship between fatty acid structure and operational conditions was analyzed and estab-

Stinging nettle (*Urtica dioica* L.) leaves were purchased from Institute "Dr Josif Pančić" in 2016. Leaves were dried, grounded in blender, and stored in the paper bags until further

Leaves were extracted using classic, Soxhlet, and supercritical fluid extraction methods. Classic extraction was performed by mixing 5.00 g of leaves with mixture of formaldehyde and ethanol (2:1; v/v) under vacuum at room temperature. Soxhlet extraction was performed using

critical properties (Tc = 31.1°C; pc = 73.8 bar) [36].

lished in the case of supercritical fluid extraction.

**2. Experimental section**

**2.2. Extraction procedures**

**2.1. Plant material**

processing.

Previously conducted studies showed that stinging nettle contains wide diversity of biologically active compounds. There are essential oils and terpenoids as the main components [9, 10], carotenoids [4, 9, 11], fatty acids [9, 11, 12], different phenolic and polyphenolic compounds [1, 3, 9, 13, 14], essential amino acids, chlorophyll, vitamin C, vitamin K, vitamins of B series, tannins, carbohydrates, sterols, polysaccharides, isolectins [4, 9, 11, 15], and minerals [9, 16, 17]. The most important carotenoids are β-carotene, neoxanthin, lycopene, and lutein [11]; fatty acids are palmitic and *cis*-9,12 linoleic and α-linolenic acids [9, 11, 12], while iron has been marked as the most important mineral [9].

Fatty acids or generally fats are presented in many different forms such as saturated, monounsaturated, unsaturated, omega-3, omega-6, trans, or partially hydrogenated fats. These terms are widely presented in media regarding the health subject. Fats represent the most efficient way of storing excess energy. Besides this role, fats represent building blocks of lipid membranes (in the form of phospholipids), play significant roles in maintaining proper function of brain, and participate in process of signal transduction (in the form of diacylglycerols). Certain fats such as linoleic and arachidonic are essential due to inability of human body to synthetize them. Thus, their presence is very important because their involvement in biosynthesis of eicosanoids. This class of biomolecules is further involved in wide range of processes in organism such as platelet aggregation, anti-inflammatory response, allergic reactions, etc. Polyunsaturated fatty acids decrease LDL and cholesterol levels, while saturated acids increase both [18]. It has been found that n-3 and n-6 fatty acids expressed anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic, and vasodilatory properties [19]. The n-3 fatty acids proved to be secondary preventers of several diseases and disorders such as coronary heart disease, hypertension, type 2 diabetes, renal disease, rheumatoid arthritis, ulcerative colitis, Crohn's disease, and chronic obstructive pulmonary disease [19] but also show effects in decreasing the risk of heart disease and cancer [20, 21].

Extraction represents the most common way of isolation of desired compound from the mixture, natural sources, or other matrices. Generally, extraction techniques may be divided into conventional and nonconventional. Usual conventional techniques are classic extraction and Soxhlet extraction. Such techniques usually applied toxic and environmental non-friendly solvents [22]. Due to such drawbacks, nonconventional techniques such as ultrasound-assisted, microwaveassisted, and subcritical water and supercritical fluid extractions have been developed.

Ultrasound (20 KHz–100 MHz) and microwave (300MHz–300 GHz) penetrate through medium causing characteristic effects which enhance mass transfer. Ultrasound creates compression and expansion, thus producing cavitation [23]. Microwave on the other hand creates uniform heating as a consequence of medium resistance to ion flow. This effect is in close correlation with ionic conduction and dipole rotation mechanisms [23–25]. Supercritical water extraction relies on application of pressure to maintain water in liquid state during its heating above boiling point. Such state causes decrease in dielectric constant of water together with the polarity. Thus, polarity may, under certain conditions, become close to polarity of methanol [26–28].

Supercritical fluid extraction is a suitable substitution for conventional approaches such as hydrodistillation, steam distillation, and solvent extraction [29]. Techniques have started its commercial application during the 1980s [30]. Industrial scale of this technique found its application in processes such as decaffeination of green coffee beans and black tea leaves, production of hop extracts, isolation of essential oils, oleoresins and flavoring compounds from natural sources, extraction and fractionation of edible oils, and removal the pesticides from plant materials [31, 32]. This technique relies on application of fluids in their supercritical state. Such state can be achieved at pressures and temperatures above critical ones for given fluid. When this state has been achieved, fluid expressed properties between those characteristic for gas and liquid state [33, 34]. Density of fluid is similar to the density of liquids, while viscosity is similar to the values for gas. Behavior of two important factors influences the diffusivity which ranges between values for liquid and gas state. This results in better transport properties [34]. It should be mentioned that most important characteristic of fluids in this state is the ability to modulate their properties throughout modulation of density of fluid itself. This may be achieved by changing the pressure and temperature of system. Such changes in density influence directly the solubility of desired compounds in the fluid [35]. Carbon dioxide is the most commonly used fluid for this technique due to its nontoxic and nonexplosive properties, low price, availability, easy removable from extracts, and moderate critical properties (Tc = 31.1°C; pc = 73.8 bar) [36].

Due to importance of fatty acids for proper function of human organism, it is also important to establish their occurrence in nature and possibility for utilization. The main goal of this research was to isolate and establish fatty acid profile in stinging nettle leaves. Different extraction techniques were used in order to compare their efficiency for isolation of fatty acids, while GC-FID technique was applied for their quantification. From the presented results, relationship between fatty acid structure and operational conditions was analyzed and established in the case of supercritical fluid extraction.

## **2. Experimental section**

## **2.1. Plant material**

been used for treatment of flailing arthritis or paralytic limbs, stimulation of circulation, and warming the joints and extremities [2]. There are wide ranges of studies which have dealt with biological activity of this plant. They have proved antioxidant, antimicrobial, anti-inflammatory, antiulcer, and analgesic properties of plant and its extracts [2–7]. Besides application in medicine, this plant has also been used in human nutrition for a long time. It has been harvested commercially for high content of chlorophylls, which has been used as

Previously conducted studies showed that stinging nettle contains wide diversity of biologically active compounds. There are essential oils and terpenoids as the main components [9, 10], carotenoids [4, 9, 11], fatty acids [9, 11, 12], different phenolic and polyphenolic compounds [1, 3, 9, 13, 14], essential amino acids, chlorophyll, vitamin C, vitamin K, vitamins of B series, tannins, carbohydrates, sterols, polysaccharides, isolectins [4, 9, 11, 15], and minerals [9, 16, 17]. The most important carotenoids are β-carotene, neoxanthin, lycopene, and lutein [11]; fatty acids are palmitic and *cis*-9,12 linoleic and α-linolenic acids [9, 11, 12], while iron has been

Fatty acids or generally fats are presented in many different forms such as saturated, monounsaturated, unsaturated, omega-3, omega-6, trans, or partially hydrogenated fats. These terms are widely presented in media regarding the health subject. Fats represent the most efficient way of storing excess energy. Besides this role, fats represent building blocks of lipid membranes (in the form of phospholipids), play significant roles in maintaining proper function of brain, and participate in process of signal transduction (in the form of diacylglycerols). Certain fats such as linoleic and arachidonic are essential due to inability of human body to synthetize them. Thus, their presence is very important because their involvement in biosynthesis of eicosanoids. This class of biomolecules is further involved in wide range of processes in organism such as platelet aggregation, anti-inflammatory response, allergic reactions, etc. Polyunsaturated fatty acids decrease LDL and cholesterol levels, while saturated acids increase both [18]. It has been found that n-3 and n-6 fatty acids expressed anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic, and vasodilatory properties [19]. The n-3 fatty acids proved to be secondary preventers of several diseases and disorders such as coronary heart disease, hypertension, type 2 diabetes, renal disease, rheumatoid arthritis, ulcerative colitis, Crohn's disease, and chronic obstructive pulmonary disease [19] but also show effects

Extraction represents the most common way of isolation of desired compound from the mixture, natural sources, or other matrices. Generally, extraction techniques may be divided into conventional and nonconventional. Usual conventional techniques are classic extraction and Soxhlet extraction. Such techniques usually applied toxic and environmental non-friendly solvents [22]. Due to such drawbacks, nonconventional techniques such as ultrasound-assisted, microwave-

Ultrasound (20 KHz–100 MHz) and microwave (300MHz–300 GHz) penetrate through medium causing characteristic effects which enhance mass transfer. Ultrasound creates compression and expansion, thus producing cavitation [23]. Microwave on the other hand creates uniform heating as a consequence of medium resistance to ion flow. This effect is in close correlation with ionic conduction and dipole rotation mechanisms [23–25]. Supercritical water extraction

assisted, and subcritical water and supercritical fluid extractions have been developed.

coloring agent (E140) in food and medical products [8].

in decreasing the risk of heart disease and cancer [20, 21].

marked as the most important mineral [9].

70 Carboxylic Acid - Key Role in Life Sciences

Stinging nettle (*Urtica dioica* L.) leaves were purchased from Institute "Dr Josif Pančić" in 2016. Leaves were dried, grounded in blender, and stored in the paper bags until further processing.

## **2.2. Extraction procedures**

Leaves were extracted using classic, Soxhlet, and supercritical fluid extraction methods. Classic extraction was performed by mixing 5.00 g of leaves with mixture of formaldehyde and ethanol (2:1; v/v) under vacuum at room temperature. Soxhlet extraction was performed using 5.00 g of leaves and 100.00 mL of petrol ether. Process was conducted in automated Soxhlet apparatus Soxtherm S306 (C. Gerhardt, Germany) at 150°C, under constant pressure for 2 h.

**Fatty acid Content (mg/g)**

**Table 1.** Fatty acid profile of classic and Soxhlet extracts.

C6:0 / 0.667 C8:0 / 4.925 C10:0 / 4.677 C12:0 0.202 45.639 C14:0 0.456 20.656 C14:1 0.085 0.610 C15:0 / 0.722 C15:1 / 0.375 C16:0 3.022 40.523 C16:1 0.158 0.548 C17:0 0.054 0.568 C17:1 0.072 0.288 C18:0 0.493 19.045 C18:1 *cis*-Δ9 0.486 28.773 C18:2 *cis*-Δ9,12 2.775 6.579 C20:0 1.036 11.512 C18:3 *cis*-Δ9,12,15 3.368 2.232 C21:0 0.137 1.075 C20:2 *cis*-Δ11,14 0.423 1.663 C22:0 0.890 13.385 C20:4 *cis*-Δ5,8,11,14 0.087 0.935 C23:0 0.174 0.400 C22:2 *cis*-Δ13,16 0.218 0.775 C24:0 0.644 10.554 C20:5 *cis*-Δ5,8,11,14,17 / 0.685 C24:1 0.251 1.096 SFAs 7.108 173.348 UFAs 7.923 44.559 MUFAs 1.052 31.690 PUFAs 6.871 12.869 Total 15.031 218.907 SFA:UFA ratio 0.90 3.91

**Classic extraction Soxhlet extraction**

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Supercritical fluid extraction was conducted using previously described apparatus (HPEP, NOVA-Swiss, Effretikon, Switzerland) [36, 37]. Main parts of this plant are gas cylinder with carbon dioxide, diaphragm-type compressor, extractor vessel with heating jacket, separator, pressure control valve, temperature regulation system, and regulation valves. Extractions were performed at three different pressure (100, 200, and 300 bar) and two different temperature (40 and 60°C) levels, while CO2 flow rate was maintained constant (0.20 kg/h).

## **2.3. Sample preparation and fatty acid analysis**

Extracts obtained using above-described techniques were further prepared for analysis using method described elsewhere [9]. Basically, fatty acids were extracted from prepared extracts using hexane, then hydrolyzed from their natural forms, and esterified using methanolic solution of KOH.

Analysis was conducted using Agilent 7890A gas chromatograph coupled with FID detector and CP-Sil 88 column (100 m × 0.25 mm × 0.20 μm), with nitrogen flow rate of 1 mL/min, applying previously described analytical method [9]. Nitrogen flow rate in detector was 25 mL/min, and air flow was 400 mL/min, while hydrogen flow rate was 30 mL/min. Oven temperature program was as follows: initial temperature 80°C (0.5 min), then linear increase of 4°C/min up to 220°C (4 min), and linear increase of 4°C/min up to 240°C (10 min). Temperatures of injector and detector were 240°C and 270°C, respectively. Standard solution for calibration was prepared by dissolving standard compounds in hexane in concentration range of 2–120 μg/mL. Limit of detection and recovery was 0.011–0.032% and 83–126%, respectively. Fatty acid contents were expressed as mg of fatty acid per g of extract (mg/g).

## **3. Results and discussion**

## **3.1. Fatty acid contents in classic and Soxhlet extracts**

Fatty acid profile of extracts obtained using classic and Soxhlet approaches is presented in **Table 1**, while chromatograms are given in **Figure 1**. According to their molecular structure, fatty acids (FAs) were divided into saturated (SFAs) and unsaturated (UFAs). UFAs were further divided into monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids.

Presented results indicated that automatic Soxhlet approach was a betted solution for isolation of fatty acids. Fatty acid yield in Soxhlet extract was 14.6-fold higher than yield of classic approach. Classic approach was proved to be better for isolation of UFAs, while SFAs dominated over UFAs in Soxhlet extract. Major FAs in classic extracts were C18:3 (3.368 mg/g), C16:0 (3.022 mg/g), C18:2 (2.775 mg/g), and C20:0 (1.036 mg/g). Domination of those three acids was earlier confirmed by several studies [9, 11, 12]. However, Soxhlet extract showed different profiles where C12:0 (45.639 mg/g) and C16:0 (40.523 mg/g) were major FAs followed by C18:1 (28.773 mg/g), 14:0 (20.656 mg/g), C18:0 (19.045 mg/g), C22:0 (13.385 mg/g), C20:0 (11.512 mg/g), and C24:0 (10.554 mg/g).

**Table 1.** Fatty acid profile of classic and Soxhlet extracts.

5.00 g of leaves and 100.00 mL of petrol ether. Process was conducted in automated Soxhlet apparatus Soxtherm S306 (C. Gerhardt, Germany) at 150°C, under constant pressure for 2 h. Supercritical fluid extraction was conducted using previously described apparatus (HPEP, NOVA-Swiss, Effretikon, Switzerland) [36, 37]. Main parts of this plant are gas cylinder with carbon dioxide, diaphragm-type compressor, extractor vessel with heating jacket, separator, pressure control valve, temperature regulation system, and regulation valves. Extractions were performed at three different pressure (100, 200, and 300 bar) and two different tempera-

Extracts obtained using above-described techniques were further prepared for analysis using method described elsewhere [9]. Basically, fatty acids were extracted from prepared extracts using hexane, then hydrolyzed from their natural forms, and esterified using methanolic

Analysis was conducted using Agilent 7890A gas chromatograph coupled with FID detector and CP-Sil 88 column (100 m × 0.25 mm × 0.20 μm), with nitrogen flow rate of 1 mL/min, applying previously described analytical method [9]. Nitrogen flow rate in detector was 25 mL/min, and air flow was 400 mL/min, while hydrogen flow rate was 30 mL/min. Oven temperature program was as follows: initial temperature 80°C (0.5 min), then linear increase of 4°C/min up to 220°C (4 min), and linear increase of 4°C/min up to 240°C (10 min). Temperatures of injector and detector were 240°C and 270°C, respectively. Standard solution for calibration was prepared by dissolving standard compounds in hexane in concentration range of 2–120 μg/mL. Limit of detection and recovery was 0.011–0.032% and 83–126%, respectively. Fatty acid contents were

Fatty acid profile of extracts obtained using classic and Soxhlet approaches is presented in **Table 1**, while chromatograms are given in **Figure 1**. According to their molecular structure, fatty acids (FAs) were divided into saturated (SFAs) and unsaturated (UFAs). UFAs were further divided into monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids.

Presented results indicated that automatic Soxhlet approach was a betted solution for isolation of fatty acids. Fatty acid yield in Soxhlet extract was 14.6-fold higher than yield of classic approach. Classic approach was proved to be better for isolation of UFAs, while SFAs dominated over UFAs in Soxhlet extract. Major FAs in classic extracts were C18:3 (3.368 mg/g), C16:0 (3.022 mg/g), C18:2 (2.775 mg/g), and C20:0 (1.036 mg/g). Domination of those three acids was earlier confirmed by several studies [9, 11, 12]. However, Soxhlet extract showed different profiles where C12:0 (45.639 mg/g) and C16:0 (40.523 mg/g) were major FAs followed by C18:1 (28.773 mg/g), 14:0 (20.656 mg/g), C18:0 (19.045 mg/g), C22:0 (13.385 mg/g), C20:0

flow rate was maintained constant (0.20 kg/h).

ture (40 and 60°C) levels, while CO2

72 Carboxylic Acid - Key Role in Life Sciences

solution of KOH.

**2.3. Sample preparation and fatty acid analysis**

expressed as mg of fatty acid per g of extract (mg/g).

**3.1. Fatty acid contents in classic and Soxhlet extracts**

**3. Results and discussion**

(11.512 mg/g), and C24:0 (10.554 mg/g).

Tuning these two parameters allows higher level of selectivity throughout influence on density of supercritical fluid and vapor pressure of solutes. Results of fatty acids GC analysis are

C6:0 0.376 0.213 0.219 / 0.208 / C10:0 0.190 / / / / / C11:0 0.128 / / / / / C12:0 0.209 0.216 0.178 0.158 0.175 0.228 C13:0 0.211 0.181 0.178 / 0.143 / C14:0 3.859 1.697 2.150 1.573 1.968 1.523 C14:1 1.155 0.860 0.659 0.414 0.611 0.483 C15:0 0.204 0.151 0.173 0.404 / 0.165 C15:1 0.185 0.142 0.612 / 0.376 0.338 C16:0 8.657 6.616 8.010 7.102 6.053 6.331 C16:1 1.075 0.588 0.432 0.702 0.497 0.556 C17:0 0.350 0.294 0.148 0.763 0.192 0.235 C17:1 0.553 0.239 0.329 0.250 0.161 0.171 C18:0 1.996 1.527 1.799 2.279 1.388 1.631 C18:1 *trans*-Δ9 / 0.126 / / / / C18:1 *cis*-Δ9 2.130 1.565 1.640 1.072 1.426 1.297 C18:2 *cis*-Δ9,12 10.900 7.746 9.047 7.347 7.216 7.240 C20:0 3.851 3.986 7.734 13.934 4.286 10.107 C18:3 *cis*-Δ6,9,12 0.431 0.276 / 0.160 0.266 /

**1 2 3 4 5 6**

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**Sample Pressure (bar) Temperature (°C)**

1 100 40 2 100 60 3 200 40 4 200 60 5 300 40 6 300 60

**Table 2.** Operational parameters of supercritical fluid extraction.

**Fatty acid Sample/content (mg/g)**

presented in **Table 3**.

**Figure 1.** Chromatogram classic extract (A) and Soxhlet extract (B).

Besides the abovementioned differences, it might be also noticed that several fatty acids were detected only in Soxhlet extract (C6:0, C8:0, C10:0, C15:0, C15:1, and C20:5). Also, content of C18:3 PUFA was higher in classic extract. This might be explained with stronger interaction among C18:3 acid and ethanol, which was also previously reported [38].

#### **3.2. Fatty acid profile of SFE extracts**

Supercritical fluid extraction was conducted using three different levels of pressure and two different levels of temperature (**Table 2**).

Tuning these two parameters allows higher level of selectivity throughout influence on density of supercritical fluid and vapor pressure of solutes. Results of fatty acids GC analysis are presented in **Table 3**.

**Table 2.** Operational parameters of supercritical fluid extraction.

Besides the abovementioned differences, it might be also noticed that several fatty acids were detected only in Soxhlet extract (C6:0, C8:0, C10:0, C15:0, C15:1, and C20:5). Also, content of C18:3 PUFA was higher in classic extract. This might be explained with stronger interaction

Supercritical fluid extraction was conducted using three different levels of pressure and two

among C18:3 acid and ethanol, which was also previously reported [38].

**3.2. Fatty acid profile of SFE extracts**

74 Carboxylic Acid - Key Role in Life Sciences

different levels of temperature (**Table 2**).

**Figure 1.** Chromatogram classic extract (A) and Soxhlet extract (B).

was obvious that higher pressure and temperature levels were better for isolation of SFAs. In the case of UFAs, situation was completely different. The highest yield of UFAs was obtained in sample 1 (100 bar, 40°C), while the lowest was observed at 200 bar and 60°C (sample 4). It might be concluded that lower pressure and temperature levels were better for isolation of UFAs. This was also the same for both MUFAs and PUFAs. Such conclusions might be supported by SFA/UFA ratio, where the highest value was obtained in sample 4 (1.51) and the

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Previous investigations of relationship between structure and solubility of organic compounds in supercritical carbon dioxide showed that length of carbon chain and number of double bonds influences their miscibility in supercritical fluids. Thus, increase in carbon number causes decrease in miscibility. Introduction of double bonds showed same effect, but unsaturation expressed favorable effect where alkenes were more miscible than alkanes [40]. Same trends stand for fatty acids. With increasing in number of carbon atoms and molecular weight miscibility decreases, while introduction of double bonds decrease melting point of fatty acids and this increases their miscibility [41, 42]. These parameters together with abovementioned influence of pressure and temperature ensured presented variations in fatty acid profiles (**Table 3**). C6:0 acid was not quantified in samples 4 and 6. Comparing with its occurrence in samples 3 and 5, it might be concluded that increasing in temperature expressed negative effect on this acid. Same tendency was noticed in the case of C13:0 acid. C10:0 and C11:0 acids were quantified only in sample 1 (100 bar, 40°C). Negative influence of both pressure and temperature on miscibility of this fatty acid was clearly obvious. C15:0 acid was not quantified only in sample 5. C21:0 acid was not observed in sample 2, while C23:0 was not detected in sample 3. All other saturated acids (C12:0, C14:0, C16:0, C17:0 C18:0, C20:0, C22:0, and C24:0) were

lowest in samples 1 and 2 (0.42). Chromatogram of sample 3 is presented in **Figure 2**.

quantified in all samples.

**Figure 2.** Chromatogram of sample 3 obtained at 200 bar and 40°C.

**Table 3.** Fatty acid profile of SFE extracts.

Pressure and temperature are considered to be the most influential parameters in SFE. Generally, pressure exhibited positive influence on extraction yield due to its positive influence on density of supercritical fluid. On the other hand, temperature exhibits dual effect as a result of combination of two variables: density and vapor pressure. Density decreases with temperature causing the decreasing in solubility. Vapor pressure increases with temperature, thus increasing the solubility in the same time [30, 39]. Pressure at the point of inversion of those two variables is known as crossover pressure or crossover point [30].

Influence of all abovementioned effects might be noticed in the case of fatty acid profile presented in **Table 3**. The highest yield of FAs was observed at 200 bar and 40°C (sample 3), while the lowest was at 100 bar and 60°C (sample 2). At both temperature levels, similar tendency of FAs yield might be noticed. Yields increased from 100 to 200 bar and then decreased at 300 bar. SFA yields showed same tendency, but the highest was observed in sample 4 (200 bar, 60°C), while the lowest was noticed in sample 2 (100 bar, 60°C). From the presented results, it was obvious that higher pressure and temperature levels were better for isolation of SFAs. In the case of UFAs, situation was completely different. The highest yield of UFAs was obtained in sample 1 (100 bar, 40°C), while the lowest was observed at 200 bar and 60°C (sample 4). It might be concluded that lower pressure and temperature levels were better for isolation of UFAs. This was also the same for both MUFAs and PUFAs. Such conclusions might be supported by SFA/UFA ratio, where the highest value was obtained in sample 4 (1.51) and the lowest in samples 1 and 2 (0.42). Chromatogram of sample 3 is presented in **Figure 2**.

Previous investigations of relationship between structure and solubility of organic compounds in supercritical carbon dioxide showed that length of carbon chain and number of double bonds influences their miscibility in supercritical fluids. Thus, increase in carbon number causes decrease in miscibility. Introduction of double bonds showed same effect, but unsaturation expressed favorable effect where alkenes were more miscible than alkanes [40]. Same trends stand for fatty acids. With increasing in number of carbon atoms and molecular weight miscibility decreases, while introduction of double bonds decrease melting point of fatty acids and this increases their miscibility [41, 42]. These parameters together with abovementioned influence of pressure and temperature ensured presented variations in fatty acid profiles (**Table 3**).

C6:0 acid was not quantified in samples 4 and 6. Comparing with its occurrence in samples 3 and 5, it might be concluded that increasing in temperature expressed negative effect on this acid. Same tendency was noticed in the case of C13:0 acid. C10:0 and C11:0 acids were quantified only in sample 1 (100 bar, 40°C). Negative influence of both pressure and temperature on miscibility of this fatty acid was clearly obvious. C15:0 acid was not quantified only in sample 5. C21:0 acid was not observed in sample 2, while C23:0 was not detected in sample 3. All other saturated acids (C12:0, C14:0, C16:0, C17:0 C18:0, C20:0, C22:0, and C24:0) were quantified in all samples.

**Figure 2.** Chromatogram of sample 3 obtained at 200 bar and 40°C.

Pressure and temperature are considered to be the most influential parameters in SFE. Generally, pressure exhibited positive influence on extraction yield due to its positive influence on density of supercritical fluid. On the other hand, temperature exhibits dual effect as a result of combination of two variables: density and vapor pressure. Density decreases with temperature causing the decreasing in solubility. Vapor pressure increases with temperature, thus increasing the solubility in the same time [30, 39]. Pressure at the point of inversion of those two vari-

**1 2 3 4 5 6**

C20:1 *cis*-Δ11 0.159 0.152 / 0.184 0.174 / C18:3 *cis*-Δ9,12,15 38.564 28.943 24.186 20.504 26.235 22.375 C21:0 0.301 / 17.809 1.483 0.578 0.755 C20:2 *cis*-Δ11,14 3.872 1.364 2.258 1.256 1.794 1.477 C22:0 1.967 1.844 5.945 12.577 3.078 10.638 C20:3 *cis*-Δ8,11,14 0.244 0.292 0.138 0.125 0.447 0.129 C20:3 *cis*-Δ11,14,17 / 0.336 / / / 0.719 C20:4 *cis*-Δ5,8,11,14 0.507 0.277 / 0.294 0.338 0.256 C23:0 1.634 0.767 / 0.399 0.821 0.539 C22:2 *cis*-Δ13,16 / / 0.615 / / / C24:0 1.146 0.950 3.590 8.634 2.923 5.768 C20:5 *cis*-Δ5,8,11,14,17 / 0.603 / / 0.899 / C24:1 *cis*-Δ15 / / 1.263 / / / C22:6 *cis*-Δ7,4,10,13,16,19 0.287 0.165 / 0.261 0.250 / SFAs 25.079 18.442 47.933 49.306 21.813 37.920 UFAs 60.062 43.647 41.179 32.569 40.690 35.041 MUFAs 5.257 3.672 4.935 2.622 3.245 2.845 PUFAs 54.805 40.002 36.244 29.947 37.445 32.196 Total 85.141 62.116 89.112 81.875 62.503 72.961 SFA/UFA ratio 0.42 0.42 1.16 1.51 0.54 1.08

Influence of all abovementioned effects might be noticed in the case of fatty acid profile presented in **Table 3**. The highest yield of FAs was observed at 200 bar and 40°C (sample 3), while the lowest was at 100 bar and 60°C (sample 2). At both temperature levels, similar tendency of FAs yield might be noticed. Yields increased from 100 to 200 bar and then decreased at 300 bar. SFA yields showed same tendency, but the highest was observed in sample 4 (200 bar, 60°C), while the lowest was noticed in sample 2 (100 bar, 60°C). From the presented results, it

ables is known as crossover pressure or crossover point [30].

**Table 3.** Fatty acid profile of SFE extracts.

**Fatty acid Sample/content (mg/g)**

76 Carboxylic Acid - Key Role in Life Sciences

Comparing the yields for saturated FAs under isothermal conditions revealed existence of two trends: up to C18:0 acid yield decreased with pressure increasing at 40°C. After C18:0 acid, yield increased up to 200 bar and then decreased. On the other hand, at 60°C yield increased at first and then decreased. Changes in temperature under isobaric conditions influenced differently depending on fatty acid. At 100 bar yields decreased with increasing in temperature with the exception of C12:0, C20:0, and C23:0 where yield increased with the temperature. At 200 bar yields dropped except in the case of C15:0, C17:0, C18:0, C20:0, C22:0, C23:0, and C24:0. It might be noticed that more acids reached their maximal yield at higher temperature then in the case of 100 bar. At 300 bar yields decreased only in the case of C6:0, C13:0, C14:0, and C23:0. Such behavior might be explained with previously mentioned dual effect of temperature.

**4. Conclusion**

fatty acids in this particular case.

Saša Đurović1,2\*, Saša Šorgić3

, Saša Popov3

1 Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

2 Institute of General and Physical Chemistry, Belgrade, Serbia

\*Address all correspondence to<EMAIL_ADDRESS>
3 Oneological Laboratory, Vršac, Serbia

, Marija Radojković1

[1] Orčić D, Francišković M, Bekvalac K, Svirčev E, Beara I, Lesjak M, et al. Quantitative determination of plant phenolics in *Urtica dioica* extracts by high-performance liquid

and Zoran Zeković1

**Author details**

**References**

Three different extraction techniques were applied for isolation of fatty acids from leaves of stinging nettle. Results showed that automated Soxhlet approach provided the highest yield of fatty acids, while classic extraction achieved the lowest one. Comparison of those two conventional techniques revealed domination of certain saturated and unsaturated fatty acids. In the case of classic extraction, C16:0, C20:0, C18:2, and C18:3 were dominant compounds, while in the case of Soxhlet extraction, main acids were C12:0, C14:0, C16:0, C18:0, C18:1, C20:0, C22:0, and C24:0. It might be noticed that saturated acids absolutely dominated in this sample (SFA/UFA ratio was 3.91). Similar results were obtained in the case of SFE. Dominant acids were C16:0, C18:0, C18:1, C18:2, C20:0, C18:3, C21:0, C20:2, C22:0, and C24:0, but their content was in close connection with operational conditions, that is, pressure and temperature.

Isolation and GC Analysis of Fatty Acids: Study Case of Stinging Nettle Leaves

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79

Investigation of influence of operational conditions on fatty acid profile revealed complex connection. The highest total yield was obtained in sample 3, followed by sample 1. Both samples achieved high yield at lower temperature level. Under isothermal condition pressure changes influenced differently. At both temperature levels, yield of SFAs increased up to 200 bar and then decreased. Yield of MUFAs decreased with pressure increasing at lower temperature but increase at higher temperature level and 300 bar. Yield of PUFAs decreased up to 200 bar and then increased at both temperature levels. Isobaric processes showed that temperature influenced negatively on yield of SFAs at 100 bar. At higher pressures, temperature exhibits positive effect, which was explained by changes in domination of two variables, that is, density and vapor pressure. On the other hand, temperature increasing negatively influenced yield of UFAs, MUFAs, and PUFAs. Results showed that lower pressure and temperature levels were most beneficial conditions for isolation of unsaturated fats. To conclude, careful choice of operational condition might result in favored isolation of desired group of

Effects of SFE conditions on yields of unsaturated acids will be considered separately for monounsaturated and polyunsaturated fatty acids. Results showed that *trans*-C18:1 acid was quantified only in sample 2, while 24:1 acid was quantified in sample 3. C15:1 acid was not detected in sample 4, while C20:1 acid was not quantified in samples 3 and 6. C14:1, C17:1, and *cis*-C18:1 acids were quantified in all samples. Isothermal conditions again showed dual effect. At 40°C yield constantly decreased with pressure increasing for C14:1, C17:1, and *cis*-C18:1 acids. In the case of isothermal process, at 60°C, yields reached their maximal values at 200 or 300 bar depending on acid. Isobaric conditions at 100 bar showed that yields dropped with increasing in temperature in all cases except for *trans*-C18:1 acid. At 200 bar trends were the same with the exception of C16:1 and C20:1 acid whose yields increased with temperature. In the case of 300 bar, yields of C16:1 and C17:1 decreased with increasing in temperature.

Polyunsaturated fatty acids were also detected and quantified in all samples. Dominant PUFA was C18:3 *cis*-Δ9,12,15 acid, which was the most abundant acid in all samples. C18:3 *cis*-Δ6,9,12 was not quantified in samples 3 and 6. C20:3 *cis*-Δ11,14,17 acid was detected only in samples 2 and 6, and C22:2 was found only in sample 3, while C20:5 was quantified only in sample 2. C20:4 was not found only in sample 3. Almost all PUFAs reached their maximal yields at 100 bar and 40°C (sample 1) with the exception of 20:3 *cis*-Δ8,11,14, 20:3 *cis*-Δ11,14,17, and C22:2 acids. They achieved their maximal yields in samples 5, 6, and 3, respectively. Both 20:3 acids achieved their maximal yield at 300 bar but at different temperature level. This might be explained with different positions of double bonds in their structures.

Deeper investigation showed that under isothermal conditions at 40°C, yields of most of compounds decreased up to 200 bar and then increased at 300 bar. Exceptions were C18:2 and 20:2 acids whose yields decreased at all three pressure levels. At 60°C, yields of C18:2 and C18:3 *cis*-Δ6,9,12 constantly decreased, while other acids showed different trends (yield increased or decreased up to 200 bar). Presented result showed that generally lower pressure was more beneficial for isolation of PUFAs and MUFAs (100 bar), while higher pressure is better for isolation of SFAs (200 bar). Temperature changes caused decreasing in yield of observed analytes, that is, UFAs, MUFAs, and PUFAs, at all three pressure levels (**Table 3**). They reached maximal levels at 40°C at 100 bar. Therefore, total yield of FAs showed same tendency where the highest yield was at 200 bar and 40°C followed by yield at 100 bat and 40°C. On the other hand, temperature negatively influenced SFA yield at 100 bar, but at 200 and 300 bars, yield of SFAs increased with temperature.

## **4. Conclusion**

Comparing the yields for saturated FAs under isothermal conditions revealed existence of two trends: up to C18:0 acid yield decreased with pressure increasing at 40°C. After C18:0 acid, yield increased up to 200 bar and then decreased. On the other hand, at 60°C yield increased at first and then decreased. Changes in temperature under isobaric conditions influenced differently depending on fatty acid. At 100 bar yields decreased with increasing in temperature with the exception of C12:0, C20:0, and C23:0 where yield increased with the temperature. At 200 bar yields dropped except in the case of C15:0, C17:0, C18:0, C20:0, C22:0, C23:0, and C24:0. It might be noticed that more acids reached their maximal yield at higher temperature then in the case of 100 bar. At 300 bar yields decreased only in the case of C6:0, C13:0, C14:0, and C23:0. Such behavior might be explained with previously mentioned dual effect of temperature.

Effects of SFE conditions on yields of unsaturated acids will be considered separately for monounsaturated and polyunsaturated fatty acids. Results showed that *trans*-C18:1 acid was quantified only in sample 2, while 24:1 acid was quantified in sample 3. C15:1 acid was not detected in sample 4, while C20:1 acid was not quantified in samples 3 and 6. C14:1, C17:1, and *cis*-C18:1 acids were quantified in all samples. Isothermal conditions again showed dual effect. At 40°C yield constantly decreased with pressure increasing for C14:1, C17:1, and *cis*-C18:1 acids. In the case of isothermal process, at 60°C, yields reached their maximal values at 200 or 300 bar depending on acid. Isobaric conditions at 100 bar showed that yields dropped with increasing in temperature in all cases except for *trans*-C18:1 acid. At 200 bar trends were the same with the exception of C16:1 and C20:1 acid whose yields increased with temperature. In the case of 300 bar, yields of C16:1 and C17:1 decreased with

Polyunsaturated fatty acids were also detected and quantified in all samples. Dominant PUFA was C18:3 *cis*-Δ9,12,15 acid, which was the most abundant acid in all samples. C18:3 *cis*-Δ6,9,12 was not quantified in samples 3 and 6. C20:3 *cis*-Δ11,14,17 acid was detected only in samples 2 and 6, and C22:2 was found only in sample 3, while C20:5 was quantified only in sample 2. C20:4 was not found only in sample 3. Almost all PUFAs reached their maximal yields at 100 bar and 40°C (sample 1) with the exception of 20:3 *cis*-Δ8,11,14, 20:3 *cis*-Δ11,14,17, and C22:2 acids. They achieved their maximal yields in samples 5, 6, and 3, respectively. Both 20:3 acids achieved their maximal yield at 300 bar but at different temperature level. This might be explained with

Deeper investigation showed that under isothermal conditions at 40°C, yields of most of compounds decreased up to 200 bar and then increased at 300 bar. Exceptions were C18:2 and 20:2 acids whose yields decreased at all three pressure levels. At 60°C, yields of C18:2 and C18:3 *cis*-Δ6,9,12 constantly decreased, while other acids showed different trends (yield increased or decreased up to 200 bar). Presented result showed that generally lower pressure was more beneficial for isolation of PUFAs and MUFAs (100 bar), while higher pressure is better for isolation of SFAs (200 bar). Temperature changes caused decreasing in yield of observed analytes, that is, UFAs, MUFAs, and PUFAs, at all three pressure levels (**Table 3**). They reached maximal levels at 40°C at 100 bar. Therefore, total yield of FAs showed same tendency where the highest yield was at 200 bar and 40°C followed by yield at 100 bat and 40°C. On the other hand, temperature negatively influenced SFA yield at 100 bar, but at 200 and 300 bars, yield

increasing in temperature.

78 Carboxylic Acid - Key Role in Life Sciences

different positions of double bonds in their structures.

of SFAs increased with temperature.

Three different extraction techniques were applied for isolation of fatty acids from leaves of stinging nettle. Results showed that automated Soxhlet approach provided the highest yield of fatty acids, while classic extraction achieved the lowest one. Comparison of those two conventional techniques revealed domination of certain saturated and unsaturated fatty acids. In the case of classic extraction, C16:0, C20:0, C18:2, and C18:3 were dominant compounds, while in the case of Soxhlet extraction, main acids were C12:0, C14:0, C16:0, C18:0, C18:1, C20:0, C22:0, and C24:0. It might be noticed that saturated acids absolutely dominated in this sample (SFA/UFA ratio was 3.91). Similar results were obtained in the case of SFE. Dominant acids were C16:0, C18:0, C18:1, C18:2, C20:0, C18:3, C21:0, C20:2, C22:0, and C24:0, but their content was in close connection with operational conditions, that is, pressure and temperature.

Investigation of influence of operational conditions on fatty acid profile revealed complex connection. The highest total yield was obtained in sample 3, followed by sample 1. Both samples achieved high yield at lower temperature level. Under isothermal condition pressure changes influenced differently. At both temperature levels, yield of SFAs increased up to 200 bar and then decreased. Yield of MUFAs decreased with pressure increasing at lower temperature but increase at higher temperature level and 300 bar. Yield of PUFAs decreased up to 200 bar and then increased at both temperature levels. Isobaric processes showed that temperature influenced negatively on yield of SFAs at 100 bar. At higher pressures, temperature exhibits positive effect, which was explained by changes in domination of two variables, that is, density and vapor pressure. On the other hand, temperature increasing negatively influenced yield of UFAs, MUFAs, and PUFAs. Results showed that lower pressure and temperature levels were most beneficial conditions for isolation of unsaturated fats. To conclude, careful choice of operational condition might result in favored isolation of desired group of fatty acids in this particular case.

## **Author details**

Saša Đurović1,2\*, Saša Šorgić3 , Saša Popov3 , Marija Radojković1 and Zoran Zeković1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

2 Institute of General and Physical Chemistry, Belgrade, Serbia

3 Oneological Laboratory, Vršac, Serbia

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*Edited by Georgiana Ileana Badea and Gabriel Lucian Radu*

This book is an attempt to bring together current knowledge on the role and importance of organic acids in life processes. There are lots of compounds based on the chemical nature of this functional group, which makes this class of molecules to be present in our lives starting with the human body (Krebs cycle—the core of cellular metabolism) to the products we currently use (food, medicines and cosmetics). No overall consensus is sought in this book, and the following chapters are authored by dedicated researchers presenting a diversity of applications and hypotheses concerning organic acids.

The five chapters in this book include general information on carboxylic acids and their applications in life sciences (use in organic synthesis, nanotechnology, plant physiology, plant nutrition and soil chemistry).

Published in London, UK © 2018 IntechOpen © DutchlightNetherlands / iStock

Carboxylic Acid - Key Role in Life Sciences

Carboxylic Acid

Key Role in Life Sciences

*Edited by Georgiana Ileana Badea*

*and Gabriel Lucian Radu*


**5. PDT CRC clinical challenges**

*Multidisciplinary Approach for Colorectal Cancer*

Porfimer sodium (PII) and 2-[1-hexyloxyethyl]-2-

Tetra-α-(4-carboxyphenoxy) phthalocyanine zinc

*In vivo* **PDT CRC research**

butylimide methyl ester

Metalloporphyrin Ga-4cisPtTPyP (5,10,15,20-tetrakis{cis-diammine-chloroplatinum(II)}(4-pyridyl)-porphyrinato gallium(III) hydroxide tetranitrate)

Porphyrazine platform with gadolinium (III) cation chelated by tetrapyrrole macrocycles (GdPz1 and GdPz2)

Bacteriochlorin analogues: 3-(1′-butyloxy) ethyl-3-deacetyl-bacteriopurpurin-18-N-

devinylpyropheophorbide-a (HPPH)

**Photosensitizer Remarks Ref.**

Protoporphyrin IX (PpIX) Enhanced the apoptosis in HCT116 CRC cell line

Sulfonated zinc phthalocyanine (ZnPcSmix) Within CRC DLD-1 and CaCo-2 cells the PS

δ-aminolevulinic acid (ALA) CRC cell lines SW480 and SW620 were treated

Hydrophilic bacteriochlorin (F2 BOH) PDT enabled long-term cures of BALB/c

Photosan-II (PS-II) and chloroquine Significantly reduced the tumor size in a

Redaporfin Single dose was well tolerated by male BALB/c

tumor immunity.

carcinoma cells.

tumor inhibition.

PDT controlled metastatic tumor growth in murine colon 26-HA cells and enhanced anti

localized in multiple organelles and noted significant apoptotic PDT induced cell death.

Noted interaction between p38 MAPK and caspase-9 regulated mitochondria-PDT mediated apoptosis in LoVo human colon

in sublethal doses with ALA PDT in hypoxialike conditions with cobalt chloride and noted decreases release of VEGF and significant

High tumor uptake and long-term cure within BALB/c mice bearing Colon 26 tumors.

mice with subcutaneously implanted CT26 tumors, and the cured mice rejected tumor re-inoculation 1 year after the treatment.

xenograft mice model and induced apoptotic and autophagy cell death within *in vitro*

Selective *in vivo* accumulation within murine colon carcinoma CT26 models was observed, with significant inhibition of tumor growth.

mice with subcutaneously implanted colon (CT26) tumors and PDT led to the complete tumor regression in 83% of the mice.

High tumor accumulation and almost completely inhibited tumor growth over 2 weeks in BALB/c mice bearing Colon 26

SW620 and HCT116 cells.

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

**48**

**Table 1.**

reoccurrence [35].

small-molecular inhibitor system capable of blocking any tumor survival pathways post PDT, in order to halt possible tumor reoccurrence [35]. However, in relation to fourth-generation PSS this form of PDT treatment research is limited to only being able to target and inhibit VEGFs, in order to promote PS drugs uptake and so deter the neovascularization of tumors, preventing CRC tumor metastatic spread and

*Current PDT studies which utilize different types of PS for the in vitro, in vivo or clinical treatment of CRC.*

tumors.

At the moment clinically FDA approved first and second generation PSs for PDT oncology include: Porfimer sodium (Photofrin), 5-Aminolevulinic acid

Despite the many positive features of CRC PDT, within clinical settings this form of treatment has experienced some drawbacks in relation to PS drug solubility, mode of delivery and selective tumor uptake [64, 65].

In order to ensure the maximum levels of ROS are generated during a PDT treatment, as to ensure complete tumor destruction, the highest possible concentrations of PS drugs must be able to be successfully delivered and localize in target tumor tissues [27]. Within PDT clinical settings using first and second generation PS drugs, poor outcomes and effectiveness has been noted, as only minor amounts of PS drugs are able to overcome the human bodies biological barriers and so passively accumulate (due to the EPR effect) in tumor cells, generating very low levels ROS and tumor destruction [2, 31]. Additionally, due to this passivation process sometimes PS drugs can accumulate in healthy tissues inducing unwanted PDT side effects such as patients' photosensitivity and damage to normal tissues [26].

### **Figure 3.**

*Passive PS NP drug delivery versus active targeting moiety conjugated PS NP drug delivery, which shows targeted and enhanced CRC tumor PS drug uptake for more effective PDT treatment outcomes.*

Another issue sometimes noted in clinical settings is that PS drugs have limited solubility and so tend to aggregate during administration, limiting their overall uptake and effectivity [2]. Moreover, a PS drugs concentrated subcellular localization in a tumors mitochondria, lysosomes, endoplasmic reticulum, plasma membrane etc., is of utmost importance since ROS have only a very short half-life and so will only induce effective cell death in tumor cells if they are proximately localized within these organelles [29, 32].

Thus, shortcomings such as poor solubility, bioavailability, maximum ROS generation and tumor subcellular localization targeting need to be overcome in order to ensure the effectivity of PDT [26]. Nevertheless third generation PS drug nanoparticle (NP) drug carriers are currently being investigated to ensure PS drug solubility and improved passive uptake, with functionalized active targeting abilities (e.g. overexpressed peptides), as to ensure specific uptake in tumor cells only to enhance the overall efficacy of PDT (**Figure 3**) [29, 32].