**Green Separation Techniques**

**Chapter 2**

**Provisional chapter**

**Green Separation of Bioactive Natural Products Using**

Bioactive natural products are secondary metabolites of plants and animals generated through various biological pathways. They are the main sources of new drugs, functional food and food additives. Since their contents in plant and animal tissues are extremely small compared to those of primary metabolites, the separations of bioactive principles from complex matrixes are often the inherent bottleneck in the utilization of bioactive natural products. A novel separation technique based on a liquefied mixture of solids at its eutectic compositions is presented in this chapter. The mixture can be prepared from natural primary metabolites and therefore can be considered as a green solvent. The separation of bioactive compounds (γ-oryzanol) from rice bran oil-based biodiesel using green methods with minimum energy requirement is discussed. Other applications for separations of alkaloid and phenolic compounds from their plant matrices are also presented. Different raw materials require different separation techniques due to the presence of different impurities, and the current trend is to use green methods with minimum energy requirement. This overview of recent technological advances, discussion of pertinent problems and prospect of current methodologies in the separation of bioactive natural products may provide a driving force for the development of novel

**Green Separation of Bioactive Natural Products Using** 

DOI: 10.5772/intechopen.71755

© 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,

© 2018 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.

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

Bioactive compounds of plants, also known as natural products, are produced as secondary metabolites. Unlike primary metabolites, such as carbohydrates, proteins, fats, amino acids,

**Keywords:** green separation, bioactive natural products, deep eutectic solvent, natural

**Liquefied Mixture of Solids**

**Liquefied Mixture of Solids**

Siti Zullaikah, Orchidea Rachmaniah,

Helda Niawanti and Yi Hsu Ju

http://dx.doi.org/10.5772/intechopen.71755

separation techniques.

deep eutectic solvent

**1. Introduction**

**Abstract**

Adi Tjipto Utomo, Helda Niawanti and Yi Hsu Ju

Siti Zullaikah, Orchidea Rachmaniah, Adi Tjipto Utomo,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

### **Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids Liquefied Mixture of Solids**

**Green Separation of Bioactive Natural Products Using** 

DOI: 10.5772/intechopen.71755

Siti Zullaikah, Orchidea Rachmaniah, Adi Tjipto Utomo, Helda Niawanti and Yi Hsu Ju Helda Niawanti and Yi Hsu Ju Additional information is available at the end of the chapter

Siti Zullaikah, Orchidea Rachmaniah, Adi Tjipto Utomo,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71755

#### **Abstract**

Bioactive natural products are secondary metabolites of plants and animals generated through various biological pathways. They are the main sources of new drugs, functional food and food additives. Since their contents in plant and animal tissues are extremely small compared to those of primary metabolites, the separations of bioactive principles from complex matrixes are often the inherent bottleneck in the utilization of bioactive natural products. A novel separation technique based on a liquefied mixture of solids at its eutectic compositions is presented in this chapter. The mixture can be prepared from natural primary metabolites and therefore can be considered as a green solvent. The separation of bioactive compounds (γ-oryzanol) from rice bran oil-based biodiesel using green methods with minimum energy requirement is discussed. Other applications for separations of alkaloid and phenolic compounds from their plant matrices are also presented. Different raw materials require different separation techniques due to the presence of different impurities, and the current trend is to use green methods with minimum energy requirement. This overview of recent technological advances, discussion of pertinent problems and prospect of current methodologies in the separation of bioactive natural products may provide a driving force for the development of novel separation techniques.

**Keywords:** green separation, bioactive natural products, deep eutectic solvent, natural deep eutectic solvent

#### **1. Introduction**

Bioactive compounds of plants, also known as natural products, are produced as secondary metabolites. Unlike primary metabolites, such as carbohydrates, proteins, fats, amino acids,

nucleic acids and organic acids, which are essential to perform the metabolic rules involved in the life process, they have no apparent direct functions in growth, development and reproduction. They are often differentially distributed among limited groups of plants and only present in very low quantities in plants. Though, in principle, they are inessential to life, many secondary metabolites found in plants have roles in defense against predators (herbivores, pests and pathogens), competition and facilitating the reproduction process. However, many of them still remain unknown in their functions. Previously, secondary metabolites were generally thought to be waste products of plants without apparent function. Nowadays, they represent an important source of biological active compounds which are very important for the development of food and pharmaceutical industries.

with their own unique structures and properties. Therefore, there has been an increase in the use of ILs as green solvent for the extraction, separation and purification of natural bioactive compounds [2]. However, there are concerns about the application of ILs related to the toxicity of these compounds, their potential effects on health and the environment and the high cost associ-

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

19

To overcome the drawbacks of ILs, deep eutectic solvents (DESs) have been developed. They have physicochemical properties similar to those of ILs. In addition, DESs are biodegradable, less toxic and cheaper than ILs [12]. DESs are formed from mixtures of two or more Lewis acids and bases or Bronsted-Lowry acids and bases that have the lowest freezing points compare to their starting constituents [13]. The physical structures of some DESs are similar to those of ILs. However, DESs in general are different in terms of the source of the starting

Deep eutectic solvent (DES) is a eutectic mixture of two or more compounds which has a melting point much lower than either of the individual components [6, 14, 15]. A eutectic mixture is the condition when the molar ratio of the component gives the lowest melting point as represented in **Figure 1**. DES was first introduced by Abbott et al. [14] who studied the properties of choline chloride (ChCl)/urea mixture. Both ChCl and urea have melting points of 302 and 133°C, respectively. However, at the eutectic composition (1:2 ChCl/urea molar ratio), the

Typically, DESs are mixtures of quaternary ammonium halide salts and hydrogen bond donors (HBDs). Various quaternary ammonium halide salts and HBDs which can form DESs are shown in **Figure 2**. One of the most widely used ammonium quaternary salt for DESs

ated with their synthesis and purification requirements [8, 11].

mixture melts at 12°C making it liquid at room temperature.

**Figure 1.** Schematic representation of a eutectic point on a two-component phase diagram [15].

ingredients and the chemical formation process.

**2. Eutectic solvents**

Since their content is small and different raw materials require different isolation techniques due to the presence of different impurities, the extractions of bioactive principles from complex matrixes are often the inherent bottleneck in the utilization of bioactive natural products. The extraction techniques can be classified into conventional and modern ones [1]. The conventional techniques include maceration, percolation, Soxhlet extraction and solvent extraction. They are typically characterized by long extraction time, high cost due to the requirement of large volume of solvents, low yield and the use of toxic and flammable solvents. The modern techniques include enzyme-assisted extraction, ultrasound-assisted extraction, microwaveassisted extraction, subcritical and supercritical fluid extraction and high pressure-assisted extraction. They generally have shorter extraction time, lower cost, higher purity of the extracted compounds and much better efficiency [2]. However, there are still issues associated with conventional and modern extraction techniques, including the toxicity of solvent, thermal instability, solubility and poor selectivity. In addition, the type and concentration of solvents, their moisture contents, recovery of bioactive compounds and changes of bioactive compounds during extraction due to ionization, hydrolysis, esterification and oxidation need to be considered [3]. Water can be used as solvent in the extraction of bioactive compounds. However, water is only effective to extract polar and hydrophilic bioactive compounds but less effective to extract non-polar and hydrophobic ones. In addition, impurities extracted by water become another problem for further purification steps.

Green solvents have been explored to replace traditional hazardous solvents that are used extensively in industry. To be qualified as a green solvent, a solvent should be non-toxic, non-volatile, biodegradable without generating any toxic and persistent metabolite, inflammable, recyclable, relatively cheap and available in a large quantity [4–6]. Water at sub- and supercritical conditions and CO2 at supercritical condition are examples of green solvents that have been used in industrial scale.

Recently, ionic liquids (ILs) have been developed as green solvents. Ionic liquids are molten salts, mixtures of bulky and asymmetric organic cations and organic or inorganic anions, with melting points usually below 100°C [2, 7]. They have some attractive attributes, such as non-flammable, high thermal and chemical stabilities and low vapor pressure [1, 4, 8–10]. The combination of different cations and anions makes ILs have a tunable nature, i.e. polarity and other properties with their own unique structures and properties. Therefore, there has been an increase in the use of ILs as green solvent for the extraction, separation and purification of natural bioactive compounds [2]. However, there are concerns about the application of ILs related to the toxicity of these compounds, their potential effects on health and the environment and the high cost associated with their synthesis and purification requirements [8, 11].

To overcome the drawbacks of ILs, deep eutectic solvents (DESs) have been developed. They have physicochemical properties similar to those of ILs. In addition, DESs are biodegradable, less toxic and cheaper than ILs [12]. DESs are formed from mixtures of two or more Lewis acids and bases or Bronsted-Lowry acids and bases that have the lowest freezing points compare to their starting constituents [13]. The physical structures of some DESs are similar to those of ILs. However, DESs in general are different in terms of the source of the starting ingredients and the chemical formation process.

## **2. Eutectic solvents**

nucleic acids and organic acids, which are essential to perform the metabolic rules involved in the life process, they have no apparent direct functions in growth, development and reproduction. They are often differentially distributed among limited groups of plants and only present in very low quantities in plants. Though, in principle, they are inessential to life, many secondary metabolites found in plants have roles in defense against predators (herbivores, pests and pathogens), competition and facilitating the reproduction process. However, many of them still remain unknown in their functions. Previously, secondary metabolites were generally thought to be waste products of plants without apparent function. Nowadays, they represent an important source of biological active compounds which are very important for

Since their content is small and different raw materials require different isolation techniques due to the presence of different impurities, the extractions of bioactive principles from complex matrixes are often the inherent bottleneck in the utilization of bioactive natural products. The extraction techniques can be classified into conventional and modern ones [1]. The conventional techniques include maceration, percolation, Soxhlet extraction and solvent extraction. They are typically characterized by long extraction time, high cost due to the requirement of large volume of solvents, low yield and the use of toxic and flammable solvents. The modern techniques include enzyme-assisted extraction, ultrasound-assisted extraction, microwaveassisted extraction, subcritical and supercritical fluid extraction and high pressure-assisted extraction. They generally have shorter extraction time, lower cost, higher purity of the extracted compounds and much better efficiency [2]. However, there are still issues associated with conventional and modern extraction techniques, including the toxicity of solvent, thermal instability, solubility and poor selectivity. In addition, the type and concentration of solvents, their moisture contents, recovery of bioactive compounds and changes of bioactive compounds during extraction due to ionization, hydrolysis, esterification and oxidation need to be considered [3]. Water can be used as solvent in the extraction of bioactive compounds. However, water is only effective to extract polar and hydrophilic bioactive compounds but less effective to extract non-polar and hydrophobic ones. In addition, impurities extracted by

Green solvents have been explored to replace traditional hazardous solvents that are used extensively in industry. To be qualified as a green solvent, a solvent should be non-toxic, non-volatile, biodegradable without generating any toxic and persistent metabolite, inflammable, recyclable, relatively cheap and available in a large quantity [4–6]. Water at sub- and

Recently, ionic liquids (ILs) have been developed as green solvents. Ionic liquids are molten salts, mixtures of bulky and asymmetric organic cations and organic or inorganic anions, with melting points usually below 100°C [2, 7]. They have some attractive attributes, such as non-flammable, high thermal and chemical stabilities and low vapor pressure [1, 4, 8–10]. The combination of different cations and anions makes ILs have a tunable nature, i.e. polarity and other properties

at supercritical condition are examples of green solvents that

the development of food and pharmaceutical industries.

18 Green Chemistry

water become another problem for further purification steps.

supercritical conditions and CO2

have been used in industrial scale.

Deep eutectic solvent (DES) is a eutectic mixture of two or more compounds which has a melting point much lower than either of the individual components [6, 14, 15]. A eutectic mixture is the condition when the molar ratio of the component gives the lowest melting point as represented in **Figure 1**. DES was first introduced by Abbott et al. [14] who studied the properties of choline chloride (ChCl)/urea mixture. Both ChCl and urea have melting points of 302 and 133°C, respectively. However, at the eutectic composition (1:2 ChCl/urea molar ratio), the mixture melts at 12°C making it liquid at room temperature.

Typically, DESs are mixtures of quaternary ammonium halide salts and hydrogen bond donors (HBDs). Various quaternary ammonium halide salts and HBDs which can form DESs are shown in **Figure 2**. One of the most widely used ammonium quaternary salt for DESs

**Figure 1.** Schematic representation of a eutectic point on a two-component phase diagram [15].

Another term of DESs was introduced by Gutiérrez et al. [16] as low transition temperature mixtures (LTTMs). They are the right combinations of different molar ratios between HBD and HBA, such as lactic acid/alanine = 9:1, lactic acid/ChCl = 2:1, lactic acid/histidine = 9:1, etc. The formed liquid mixtures have glass transitions instead of melting points [18]. A complete characterization of physical properties (density, viscosity, surface tension, glass transition temperature) of LTTMs, i.e. lactic acid/ChCl = 2:1, was reported by Francisco et al. [18].

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

21

Recently, Choi et al. [19] have first reported a large number of DESs which were mixtures of ChCl with any primary metabolites, e.g. sugars, sugar alcohols, organic acids and amino acids. This type of DESs is termed as natural deep eutectic solvents (NADESs). NADESs derived from major compounds always present in all microbial, mammalian and plant cells form liquid crystals. Therefore, NADESs are believed as the third liquid phase present in the cells, in addition to the already considered known phases, i.e. water and lipid [membranes] are responsible for transporting of numerous compounds with intermediate polarity in high concentration that neither dissolve in the lipid nor in the aqueous phase. Rutin, a flavonoid which is barely soluble in water, has a solubility of 0.28 mmole/mL in glucose-choline chloride-water (GCH, molar ratio = 2:5:5), thus 50–100 times higher than that in water [20], whereas, paclitaxel and ginkgolide B, which are completely water-insoluble compounds, have solubilities of 0.81 and 5.85 mg/mL, respectively, with the same NADES type [19]. In the case of macro molecules, such as DNA and starch, they also show higher solubilities in NADESs than those in water, i.e. 1.20 and 7.55 g/mL, respectively. These facts are in line with the hypothesis of the existing alternative liquid phase to water in nature of poorly water-soluble molecules including high molecular weight molecules. Though GCH (2:5:5) has polarity close to water, GCH as a NADES shows different performance than water. It indicates that NADESs have huge potential for many practical applications since they can be designed as tailor-made solvents.

**3. Extraction of bioactive compounds from rice bran oil (RBO)-based** 

Rice bran is a promising raw material for biodiesel production. It is relatively cheap, abundant and traditionally used as cattle food. The annual worldwide production of rice bran oil (RBO) could reach 8 million tons if all rice bran produced is harnessed for oil production [21]. RBO is rich in naturally occurring biologically active and antioxidant compounds, such as phytosterols, γ-oryzanol, tocopherols and tocotrienols (tocols) [22]; however, the refinery of crude RBO requires extra processing steps due to high concentrations of free fatty acids (FFA), unsaponifiable matter and dark color [21] making it uncompetitive against other edible cook-

Since RBO contains high FFA, conventional process to produce biodiesel using base catalyst is unsuitable to convert RBO into biodiesel (fatty acid methyl esters, FAME) due to the formation of soap. Several methods have been proposed to convert crude RBO into FAME, i.e. a two-step acid-catalyzed process [23], a three-step method using both acid and base catalysts [24] and a supercritical methanol method [25]. More recently, in situ process to produce biodiesel from

rice bran without any pretreatment has also been proposed by Zullaikah et al. [26].

**biodiesel by choline chloride-based deep eutectic solvent**

ing oils, such as palm oil, soybean oil and rapeseed oil.

**Figure 2.** Halide salts and HBD for DES [13].

formation is ChCl. ChCl is cheap, biodegradable, non-toxic and can be easily extracted from biomass or synthesized from fossil fuel, while the HBD could be amides (e.g. urea) [14], carboxylic acids (e.g. oxalic acid), alcohols (e.g. glycerol) [13, 16], sugars or sugar analogues, amino or organic acids and alkylsulfates or alkyl phosphates.

The main advantage of DESs over the previous generation of ILs is that they are easier and simpler to make. The solids are mixed in gentle heat until they melt, and when they cool down, they remain in the liquid form. No purification steps are required since there is no formation of new salt and the final purity is determined by the purities of the starting materials. The design of DESs is simpler and more flexible since no reaction takes place, and therefore it does not have any strict stoichiometry limitation. The interaction between HBD and hydrogen bond acceptor (HBA) will form a liquid in their relative molar composition. The behaviors and properties of DESs can be tuned by varying the HBD and its molar ratio in the mixture [13]. DESs have moderate polarity, stability and distributed negative charge like ILs, but they are biodegradable, readily available and less toxic since DESs can be based on bulk natural product such as carbohydrates (fructose, glucose, mannose, maltose and α-cyclodextrin), sugar alcohols (sorbitol) or citric acids with urea (or N,N-dimethylurea) and inorganic salts (NH<sup>4</sup> Cl and CaCl2 ) [10, 17].

Another term of DESs was introduced by Gutiérrez et al. [16] as low transition temperature mixtures (LTTMs). They are the right combinations of different molar ratios between HBD and HBA, such as lactic acid/alanine = 9:1, lactic acid/ChCl = 2:1, lactic acid/histidine = 9:1, etc. The formed liquid mixtures have glass transitions instead of melting points [18]. A complete characterization of physical properties (density, viscosity, surface tension, glass transition temperature) of LTTMs, i.e. lactic acid/ChCl = 2:1, was reported by Francisco et al. [18].

Recently, Choi et al. [19] have first reported a large number of DESs which were mixtures of ChCl with any primary metabolites, e.g. sugars, sugar alcohols, organic acids and amino acids. This type of DESs is termed as natural deep eutectic solvents (NADESs). NADESs derived from major compounds always present in all microbial, mammalian and plant cells form liquid crystals. Therefore, NADESs are believed as the third liquid phase present in the cells, in addition to the already considered known phases, i.e. water and lipid [membranes] are responsible for transporting of numerous compounds with intermediate polarity in high concentration that neither dissolve in the lipid nor in the aqueous phase. Rutin, a flavonoid which is barely soluble in water, has a solubility of 0.28 mmole/mL in glucose-choline chloride-water (GCH, molar ratio = 2:5:5), thus 50–100 times higher than that in water [20], whereas, paclitaxel and ginkgolide B, which are completely water-insoluble compounds, have solubilities of 0.81 and 5.85 mg/mL, respectively, with the same NADES type [19]. In the case of macro molecules, such as DNA and starch, they also show higher solubilities in NADESs than those in water, i.e. 1.20 and 7.55 g/mL, respectively. These facts are in line with the hypothesis of the existing alternative liquid phase to water in nature of poorly water-soluble molecules including high molecular weight molecules. Though GCH (2:5:5) has polarity close to water, GCH as a NADES shows different performance than water. It indicates that NADESs have huge potential for many practical applications since they can be designed as tailor-made solvents.

## **3. Extraction of bioactive compounds from rice bran oil (RBO)-based biodiesel by choline chloride-based deep eutectic solvent**

formation is ChCl. ChCl is cheap, biodegradable, non-toxic and can be easily extracted from biomass or synthesized from fossil fuel, while the HBD could be amides (e.g. urea) [14], carboxylic acids (e.g. oxalic acid), alcohols (e.g. glycerol) [13, 16], sugars or sugar analogues,

The main advantage of DESs over the previous generation of ILs is that they are easier and simpler to make. The solids are mixed in gentle heat until they melt, and when they cool down, they remain in the liquid form. No purification steps are required since there is no formation of new salt and the final purity is determined by the purities of the starting materials. The design of DESs is simpler and more flexible since no reaction takes place, and therefore it does not have any strict stoichiometry limitation. The interaction between HBD and hydrogen bond acceptor (HBA) will form a liquid in their relative molar composition. The behaviors and properties of DESs can be tuned by varying the HBD and its molar ratio in the mixture [13]. DESs have moderate polarity, stability and distributed negative charge like ILs, but they are biodegradable, readily available and less toxic since DESs can be based on bulk natural product such as carbohydrates (fructose, glucose, mannose, maltose and α-cyclodextrin), sugar alcohols (sorbitol)

Cl and CaCl2

) [10, 17].

amino or organic acids and alkylsulfates or alkyl phosphates.

**Figure 2.** Halide salts and HBD for DES [13].

20 Green Chemistry

or citric acids with urea (or N,N-dimethylurea) and inorganic salts (NH<sup>4</sup>

Rice bran is a promising raw material for biodiesel production. It is relatively cheap, abundant and traditionally used as cattle food. The annual worldwide production of rice bran oil (RBO) could reach 8 million tons if all rice bran produced is harnessed for oil production [21]. RBO is rich in naturally occurring biologically active and antioxidant compounds, such as phytosterols, γ-oryzanol, tocopherols and tocotrienols (tocols) [22]; however, the refinery of crude RBO requires extra processing steps due to high concentrations of free fatty acids (FFA), unsaponifiable matter and dark color [21] making it uncompetitive against other edible cooking oils, such as palm oil, soybean oil and rapeseed oil.

Since RBO contains high FFA, conventional process to produce biodiesel using base catalyst is unsuitable to convert RBO into biodiesel (fatty acid methyl esters, FAME) due to the formation of soap. Several methods have been proposed to convert crude RBO into FAME, i.e. a two-step acid-catalyzed process [23], a three-step method using both acid and base catalysts [24] and a supercritical methanol method [25]. More recently, in situ process to produce biodiesel from rice bran without any pretreatment has also been proposed by Zullaikah et al. [26].

Crude biodiesel produced from RBO using acid-catalyzed methanolysis method typically contains about 89% FAME, 4% triglycerides (TG), 4% diglycerides (DG), 0.3% monoglycerides (MG), 0.05% FFA and 2.55% bioactive compounds, mainly phytosterols and γ-oryzanol. To meet biodiesel standard as fuel (such as [27]), purification process is required to increase FAME content to at least 96.5% and decrease unreacted oil (TG, DG, MG and FFA) contents. Crude biodiesel produced from RBO through acid-catalyzed methanolysis method has different impurity compositions to those produced from edible oil (palm oil, soybean oil and rapeseed oil) using base catalyst, and therefore a different purification process is required. Besides that, since crude biodiesel from RBO contains bioactive compounds, a purification process which is able to capture those bioactive compounds will be of interest. According to Ju and Zullaikah [22], bioactive compounds were not much degraded during acid-catalyzed methanolysis. These bioactive compounds could subsequently be isolated and sold separately as high-value by-products and therefore could reduce the production cost of biodiesel.

second one was between hydrogen in ethylene glycol and Cl<sup>−</sup>

and CH<sup>2</sup>

2.271–2.474 Å. Cl−

glycol molecules.

glycol = 1:2).

show the presence of C-H, CH<sup>3</sup>

in ChCl, and the distance was

23

http://dx.doi.org/10.5772/intechopen.71755

stretching bands. Meanwhile, the N-H stretching

as anion in ChCl forms a centerpiece by interacting with five hydroxyl

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

groups, one hydroxyl group of choline cation and four hydroxyl groups from two ethylene

As a novel green solvent, a combination of mechanisms and molecular structure of DES is still unknown [31]. Therefore, FT-IR analysis was conducted in this study to determine the functional groups of DES, and the results are shown in **Figure 3** and **Table 2**. The peak in the region of 3200–3500 showed the presence of O-H groups in choline chloride and ethylene glycol-based DES which is in agreement with that reported by Aissaoui [32]. **Figure 3** describes that there is a shift of the O-H stretching band in the DES compared to that in the choline chloride and ethylene glycol. This shift is due to the electrons of oxygen that are transferred to the hydrogen bonds making the constant force lower and resulting in a change in the vibrational state. The shifting of O-H stretching vibration indicates the presence of hydrogen bonds in DES [32]. The peaks in the region of 3000-2800 on choline chloride, ethylene glycol and DES

bands overlap with the C-H vibrational bands in the region of 3000-2800 cm−1 [33]. As shown

**Figure 3.** FT-IR analysis: (a) choline chloride, (b) ethylene glycol, (c) ethaline (molar ratio of choline chloride/ethylene

DES is a promising solvent to be employed in the purification of crude biodiesel since it is inflammable, non-toxic, biodegradable and considered as a green solvent [12]. One of the most commonly used DESs for biodiesel purification process is a mixture of choline chloride (an ammonium quaternary salt) and ethylene glycol as HBD at a molar ratio of 1:2. The mixture of ethylene glycol and ChCl is called ethaline, and some physical properties of ethaline were shown in **Table 1**. Ethaline has two interactions. The first is between the chlorine anion and the hydroxyl hydrogen atom of choline, and the second is between the anion and the hydroxyl hydrogen atoms in ethylene glycol [28]. Ethaline has a strong interaction with unreacted oil (DG, MG and FFA) and unsaponifiable matter (bioactive compounds) from crude RBO-based biodiesel due to the presence of hydroxyl groups on those compounds. On the other hand, the interaction between ethaline and FAME is relatively weak since FAME has no hydroxyl group.

Based on the previous research [29], ethylene glycol interacted into each other by making hydrogen bonding in cyclical pattern and the distance of H-O bond was 1.944 Å, whereas, ChCl in crystalline structure has three bonds consisted of C-N, C-O and C-C, and the distance of each bond is 0.01 Å. ChCl is difficult to convert into liquid at room temperature due to the small distance of ChCl bonds. Based on Wagle et al. [29], DES from ChCl and ethylene glycol has two interactions of C-H-O. The first one was between oxygen from ethylene glycol and methyl proton from ChCl, and the distance was 2.146–2.440Å. The


**Table 1.** Physical properties of ethaline (molar ratio of ChCl/ethylene glycol = 1:2).

second one was between hydrogen in ethylene glycol and Cl<sup>−</sup> in ChCl, and the distance was 2.271–2.474 Å. Cl− as anion in ChCl forms a centerpiece by interacting with five hydroxyl groups, one hydroxyl group of choline cation and four hydroxyl groups from two ethylene glycol molecules.

Crude biodiesel produced from RBO using acid-catalyzed methanolysis method typically contains about 89% FAME, 4% triglycerides (TG), 4% diglycerides (DG), 0.3% monoglycerides (MG), 0.05% FFA and 2.55% bioactive compounds, mainly phytosterols and γ-oryzanol. To meet biodiesel standard as fuel (such as [27]), purification process is required to increase FAME content to at least 96.5% and decrease unreacted oil (TG, DG, MG and FFA) contents. Crude biodiesel produced from RBO through acid-catalyzed methanolysis method has different impurity compositions to those produced from edible oil (palm oil, soybean oil and rapeseed oil) using base catalyst, and therefore a different purification process is required. Besides that, since crude biodiesel from RBO contains bioactive compounds, a purification process which is able to capture those bioactive compounds will be of interest. According to Ju and Zullaikah [22], bioactive compounds were not much degraded during acid-catalyzed methanolysis. These bioactive compounds could subsequently be isolated and sold separately as high-value by-products and therefore could reduce the production cost of biodiesel.

DES is a promising solvent to be employed in the purification of crude biodiesel since it is inflammable, non-toxic, biodegradable and considered as a green solvent [12]. One of the most commonly used DESs for biodiesel purification process is a mixture of choline chloride (an ammonium quaternary salt) and ethylene glycol as HBD at a molar ratio of 1:2. The mixture of ethylene glycol and ChCl is called ethaline, and some physical properties of ethaline were shown in **Table 1**. Ethaline has two interactions. The first is between the chlorine anion and the hydroxyl hydrogen atom of choline, and the second is between the anion and the hydroxyl hydrogen atoms in ethylene glycol [28]. Ethaline has a strong interaction with unreacted oil (DG, MG and FFA) and unsaponifiable matter (bioactive compounds) from crude RBO-based biodiesel due to the presence of hydroxyl groups on those compounds. On the other hand, the interaction between ethaline and FAME is relatively weak since FAME has no hydroxyl group. Based on the previous research [29], ethylene glycol interacted into each other by making hydrogen bonding in cyclical pattern and the distance of H-O bond was 1.944 Å, whereas, ChCl in crystalline structure has three bonds consisted of C-N, C-O and C-C, and the distance of each bond is 0.01 Å. ChCl is difficult to convert into liquid at room temperature due to the small distance of ChCl bonds. Based on Wagle et al. [29], DES from ChCl and ethylene glycol has two interactions of C-H-O. The first one was between oxygen from ethylene glycol and methyl proton from ChCl, and the distance was 2.146–2.440Å. The

Melting point (K)<sup>a</sup> 207.15 Viscosity (cP)<sup>b</sup> 36 Conductivity (mS/cm)<sup>b</sup> 7.61 Density (g/cm)<sup>b</sup> 1.12 Surface tension (mN/m)<sup>b</sup> 49

**Table 1.** Physical properties of ethaline (molar ratio of ChCl/ethylene glycol = 1:2).

a [30]. b [15].

22 Green Chemistry

As a novel green solvent, a combination of mechanisms and molecular structure of DES is still unknown [31]. Therefore, FT-IR analysis was conducted in this study to determine the functional groups of DES, and the results are shown in **Figure 3** and **Table 2**. The peak in the region of 3200–3500 showed the presence of O-H groups in choline chloride and ethylene glycol-based DES which is in agreement with that reported by Aissaoui [32]. **Figure 3** describes that there is a shift of the O-H stretching band in the DES compared to that in the choline chloride and ethylene glycol. This shift is due to the electrons of oxygen that are transferred to the hydrogen bonds making the constant force lower and resulting in a change in the vibrational state. The shifting of O-H stretching vibration indicates the presence of hydrogen bonds in DES [32]. The peaks in the region of 3000-2800 on choline chloride, ethylene glycol and DES show the presence of C-H, CH<sup>3</sup> and CH<sup>2</sup> stretching bands. Meanwhile, the N-H stretching bands overlap with the C-H vibrational bands in the region of 3000-2800 cm−1 [33]. As shown

**Figure 3.** FT-IR analysis: (a) choline chloride, (b) ethylene glycol, (c) ethaline (molar ratio of choline chloride/ethylene glycol = 1:2).


**Table 2.** Wavenumber and functional group of ethaline analysis by FT-IR<sup>a</sup> .

in **Figure 3**, the stretching vibration at 2500-3100 regions in choline chloride is invisible after the formation of DES. The presence of Cl− in DES is shown at 600 and 408 cm−1. **Figure 3** also describes that the DES had a vibration pattern similar to ethylene glycol as HBD, except that the peak is at 952.01 cm−1. The peak appears on DES is in the region of 935-955 cm−1 indicating the identity of ammonium structure of DES [32]. The FT-IR analysis describes that the establishment of DES does not lead to the formation of new functional groups in the mixture.

The purification process of crude RBO by using DES is relatively simple and can be described as follows. Crude RBO-based biodiesel and DES were mixed in a stopper glass (50 mL) at a certain molar ratio (1:8). The mixture of biodiesel and DES was heated at a certain temperature (30°C) under stirring at 300 rpm. Afterwards, the mixture was let to settle for 2 h at ambient temperature so that two layers were formed. The upper layer was biodiesel (FAME)-rich phase and the bottom layer was DES-rich phase containing biodiesel impurities including bioactive compounds. The biodiesel-rich phase (upper layer) was then separated from DESrich phase by using a separator funnel. There are several factors that influence the purification process, such as extraction time, extraction temperature and molar ratio of DES/RBO-based biodiesel. However, this chapter only discusses the effect of extraction time on FAME recovery, removal of unreacted oil and bioactive compounds and the final biodiesel composition.

56.42 to 98.88% as the extraction time was increased from 15 to 240 min, while that of MG increased from 33.15 to 93.52%. The removal efficiency of FFA was lower than DG and MG, even though they have OH- group. This is probably because FFA content in RBO-based biodiesel was much lower than those of DG and MG. Since bioactive compounds in RBO, such as γ-oryzanol, phytosterols and tocols, have OH- group, they can make hydrogen bonding with DES, and their removal efficiencies were high. The removal of bioactive compounds increased

**Figure 4.** Effect of extraction time on recovery of FAME, unreacted oil and bioactive compound removal. Operation conditions, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

25

The effect of extraction time on the contents of FAME, unreacted oil and bioactive compounds is shown in **Figures 5** and **6**, respectively. FAME content increased with extraction time (**Figure 5**), while those of unreacted oil and bioactive compound content decreased with extraction time (**Figure 6**). Longer extraction time provides longer contact time between DES- and RBObased biodiesel. Therefore, more unreacted oil and bioactive compounds diffuse to DES through the formation of hydrogen bond. Since unreacted oil and bioactive compounds were removed from RBO-based biodiesel, the FAME content increased. The FAME content after 240 min purification using DES was higher than 96.5%, exceeding that specified by the European biodiesel standard [27]. FFA is undesirable in biodiesel since it causes negative impacts, such as less oxidation stability and corrosion of vital engine components. The FFA content in biodiesel is characterized by acid value. The acid value of RBO-based biodiesel produced using acid-catalyzed methanolysis method was 0.098 mg KOH/g. This acid value was already lower than the maximal acid value stated in several biodiesel standards such as EN 14214 [27] (0.5 mg KOH/g). FFA content in biodiesel after purification was decreased from 0.05 to 0.01% (**Figure 6**). This showed that the removal efficiency of FFA using DES as

from 58.91 to 91.70% with the increasing extraction time from 15 to 240 min.

**Figure 4** shows the effects of extraction time on the biodiesel recovery and the removal of unreacted oil and bioactive compounds. Extraction time is one parameter that influences liquid–liquid extraction. The unreacted oil and bioactive compounds diffuse from biodiesel-rich phase to DES-rich phase. More unreacted oil and bioactive compounds migrate to DES-rich phase with longer contact time between crude biodiesel and DES. FAME recovery {(FAME in product/FAME in sample)×100%} also increased from 63.38 to 87.31% as the extraction time was extended from 15 to 240 min as shown in **Figure 4**.

The removal efficiency of unreacted oil and bioactive compounds has similar trend with time as shown in **Figure 4**. However, the removal efficiency of each compound is different. Since TG have no OH− group, their removal efficiency was lower than the other compounds with OH− groups, such as DG, MG, FFA and bioactive compounds. TG removal efficiency was practically unaffected by extraction time. TG removal at extraction times of 15 and 240 min were 41.32 and 39.69%, respectively. However, the removal efficiencies of unreacted oil (DG, MG and FFA) and bioactive compounds increased with time. DG removal increased from

in **Figure 3**, the stretching vibration at 2500-3100 regions in choline chloride is invisible after

describes that the DES had a vibration pattern similar to ethylene glycol as HBD, except that the peak is at 952.01 cm−1. The peak appears on DES is in the region of 935-955 cm−1 indicating the identity of ammonium structure of DES [32]. The FT-IR analysis describes that the establishment of DES does not lead to the formation of new functional groups in the mixture. The purification process of crude RBO by using DES is relatively simple and can be described as follows. Crude RBO-based biodiesel and DES were mixed in a stopper glass (50 mL) at a certain molar ratio (1:8). The mixture of biodiesel and DES was heated at a certain temperature (30°C) under stirring at 300 rpm. Afterwards, the mixture was let to settle for 2 h at ambient temperature so that two layers were formed. The upper layer was biodiesel (FAME)-rich phase and the bottom layer was DES-rich phase containing biodiesel impurities including bioactive compounds. The biodiesel-rich phase (upper layer) was then separated from DESrich phase by using a separator funnel. There are several factors that influence the purification process, such as extraction time, extraction temperature and molar ratio of DES/RBO-based biodiesel. However, this chapter only discusses the effect of extraction time on FAME recovery, removal of unreacted oil and bioactive compounds and the final biodiesel composition. **Figure 4** shows the effects of extraction time on the biodiesel recovery and the removal of unreacted oil and bioactive compounds. Extraction time is one parameter that influences liquid–liquid extraction. The unreacted oil and bioactive compounds diffuse from biodiesel-rich phase to DES-rich phase. More unreacted oil and bioactive compounds migrate to DES-rich phase with longer contact time between crude biodiesel and DES. FAME recovery {(FAME in product/FAME in sample)×100%} also increased from 63.38 to 87.31% as the extraction time

The removal efficiency of unreacted oil and bioactive compounds has similar trend with time as shown in **Figure 4**. However, the removal efficiency of each compound is different. Since

 groups, such as DG, MG, FFA and bioactive compounds. TG removal efficiency was practically unaffected by extraction time. TG removal at extraction times of 15 and 240 min were 41.32 and 39.69%, respectively. However, the removal efficiencies of unreacted oil (DG, MG and FFA) and bioactive compounds increased with time. DG removal increased from

group, their removal efficiency was lower than the other compounds with

in DES is shown at 600 and 408 cm−1. **Figure 3** also

.

the formation of DES. The presence of Cl−

**Wavenumber Functional group** 3200–3500 O-H (alcohol)

1210–1150 Tertiary amine (C-N)

**Table 2.** Wavenumber and functional group of ethaline analysis by FT-IR<sup>a</sup>

1000–1350 C-C *stretch*

2845–3000 C-H *stretching*, CH<sup>2</sup> *stretching*, CH<sup>3</sup> *stretching*

496–700 C-X stretching (X = F, Br, Cl or I)

was extended from 15 to 240 min as shown in **Figure 4**.

TG have no OH−

OH−

a [33].

24 Green Chemistry

**Figure 4.** Effect of extraction time on recovery of FAME, unreacted oil and bioactive compound removal. Operation conditions, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

56.42 to 98.88% as the extraction time was increased from 15 to 240 min, while that of MG increased from 33.15 to 93.52%. The removal efficiency of FFA was lower than DG and MG, even though they have OH- group. This is probably because FFA content in RBO-based biodiesel was much lower than those of DG and MG. Since bioactive compounds in RBO, such as γ-oryzanol, phytosterols and tocols, have OH- group, they can make hydrogen bonding with DES, and their removal efficiencies were high. The removal of bioactive compounds increased from 58.91 to 91.70% with the increasing extraction time from 15 to 240 min.

The effect of extraction time on the contents of FAME, unreacted oil and bioactive compounds is shown in **Figures 5** and **6**, respectively. FAME content increased with extraction time (**Figure 5**), while those of unreacted oil and bioactive compound content decreased with extraction time (**Figure 6**). Longer extraction time provides longer contact time between DES- and RBObased biodiesel. Therefore, more unreacted oil and bioactive compounds diffuse to DES through the formation of hydrogen bond. Since unreacted oil and bioactive compounds were removed from RBO-based biodiesel, the FAME content increased. The FAME content after 240 min purification using DES was higher than 96.5%, exceeding that specified by the European biodiesel standard [27]. FFA is undesirable in biodiesel since it causes negative impacts, such as less oxidation stability and corrosion of vital engine components. The FFA content in biodiesel is characterized by acid value. The acid value of RBO-based biodiesel produced using acid-catalyzed methanolysis method was 0.098 mg KOH/g. This acid value was already lower than the maximal acid value stated in several biodiesel standards such as EN 14214 [27] (0.5 mg KOH/g). FFA content in biodiesel after purification was decreased from 0.05 to 0.01% (**Figure 6**). This showed that the removal efficiency of FFA using DES as

extraction solvent was high. This case shows that the extraction time can affect the extraction

Operation condition, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; molar ratio of RBO-based biodiesel, DES = 1:8,

.

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

The composition of biodiesel after purification using DES is compared to that specified by biodiesel standard, EN 1424 [27], as shown in **Table 3**. The ester content, acid value, MG content and DG content meet the values specified by EN 14214 [27]. However, TG content was much

so it does not interact with DES used in this experiment. This can be overcome by using a

According Niawanti and Zullaikah [34], γ-oryzanol in the upper layer (biodiesel-rich phase) was decreased with extraction time from about 4% initially to about 1% after 240 min of extraction. The lowest γ-oryzanol content of 1.18% was obtained after 240 min of extraction time. The reason of this phenomenon is that more γ-oryzanol move from RBO-based biodiesel to DES with longer extraction time. The molar ratio of biodiesel to DES also influences the removal efficiency of γ-oryzanol. The higher molar ratio of DES to RBO-based biodiesel leads to higher removal efficiency of γ-oryzanol since more γ-oryzanol molecules are bound to DES

**4. Extraction of phenolic and alkaloid compounds using natural deep** 

In recent years, many herbs and natural compounds have increasingly been receiving public interest as complementary and alternative medicines. The natural product curcumin 1,7-bis(4-hydroxy-3-methoxy phenyl)-1,6-heptadione-3,5-dione is a dietary phytochemical obtained from the dried rhizomes of the turmeric plants. It is a natural bioactive compound that has demonstrated both antioxidant and therapeutic anticancer capabilities. However, it is not yet fully used clinically due to its inherent limitations, i.e. sparing solubility in water and low bioavailability. Curcumin (C) is extracted from *Curcuma Sp*., i.e. *Curcuma longa*,

group

27

higher than those specified by EN 14214 [27]. This is because TG does not have OH−

**Property Unit Experimental value EN 14214 [27]** Ester content % (m/m) 96.65 Min 96.5 Acid value mg KOH/g 0.02 Max 0.5 MG content % (m/m) 0.02 Max 0.8 DG content % (m/m) 0.06 Max 0.2 TG content % (m/m) 3.02 Max 0.2

process of FFA in biodiesel.

**Table 3.** Composition of biodiesel after purification using DES<sup>a</sup>

240 min extraction time.

a

multiple-step separation technique.

molecules through hydrogen bonding.

**eutectic solvent**

**Figure 5.** Effect of extraction time on FAME content. Operation conditions, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

**Figure 6.** Effect of extraction time on unreacted oil and bioactive compound content. Operation conditions, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids http://dx.doi.org/10.5772/intechopen.71755 27


a Operation condition, T = 30°C; molar ratio of ChCl, ethylene glycol = 1:2; molar ratio of RBO-based biodiesel, DES = 1:8, 240 min extraction time.

**Table 3.** Composition of biodiesel after purification using DES<sup>a</sup> .

**Figure 5.** Effect of extraction time on FAME content. Operation conditions, T = 30°C; molar ratio of ChCl, ethylene

**Figure 6.** Effect of extraction time on unreacted oil and bioactive compound content. Operation conditions, T = 30°C;

molar ratio of ChCl, ethylene glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

glycol = 1:2; and molar ratio of RBO-based biodiesel, DES = 1:8.

26 Green Chemistry

extraction solvent was high. This case shows that the extraction time can affect the extraction process of FFA in biodiesel.

The composition of biodiesel after purification using DES is compared to that specified by biodiesel standard, EN 1424 [27], as shown in **Table 3**. The ester content, acid value, MG content and DG content meet the values specified by EN 14214 [27]. However, TG content was much higher than those specified by EN 14214 [27]. This is because TG does not have OH− group so it does not interact with DES used in this experiment. This can be overcome by using a multiple-step separation technique.

According Niawanti and Zullaikah [34], γ-oryzanol in the upper layer (biodiesel-rich phase) was decreased with extraction time from about 4% initially to about 1% after 240 min of extraction. The lowest γ-oryzanol content of 1.18% was obtained after 240 min of extraction time. The reason of this phenomenon is that more γ-oryzanol move from RBO-based biodiesel to DES with longer extraction time. The molar ratio of biodiesel to DES also influences the removal efficiency of γ-oryzanol. The higher molar ratio of DES to RBO-based biodiesel leads to higher removal efficiency of γ-oryzanol since more γ-oryzanol molecules are bound to DES molecules through hydrogen bonding.

## **4. Extraction of phenolic and alkaloid compounds using natural deep eutectic solvent**

In recent years, many herbs and natural compounds have increasingly been receiving public interest as complementary and alternative medicines. The natural product curcumin 1,7-bis(4-hydroxy-3-methoxy phenyl)-1,6-heptadione-3,5-dione is a dietary phytochemical obtained from the dried rhizomes of the turmeric plants. It is a natural bioactive compound that has demonstrated both antioxidant and therapeutic anticancer capabilities. However, it is not yet fully used clinically due to its inherent limitations, i.e. sparing solubility in water and low bioavailability. Curcumin (C) is extracted from *Curcuma Sp*., i.e. *Curcuma longa*, *Curcuma zedoaria* and *Curcuma manga*, a plant of the ginger family, with other two curcuminoid compounds: demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC).

*Curcuma zedoaria* that contains 1.96% ± 0.07% of dry weight of curcuminoids was kindly donated by Sari Herbal (Sukun, Malang, Indonesia). It was chosen in this study since it is traditionally believed as an anticancer medicine in Indonesia. Twelve NADESs were used as solvents for extracting curcuminoids from fine powder of *C. zedoaria*, representing different types of NADESs: (1) ionic type, consisted of organic acids, i.e. citric acid, malic acid and lactic acid, and basic compounds such choline chloride or betaine; (2) neutral type, no ionic constituent, mixed of polyalcohol, i.e. glycerol, glycine, 1–2-propanediol and sugars; (3) acidic type, consisted of neutral compounds such sugars and acidic compounds; (4) basics type, consisted of basic compounds; and the last is (5) amphoteric type, consisted of combination of amino acids and sugars, polyalcohol or acidic compounds. In the case of ionic type of NADESs, it is

O; FS-H<sup>2</sup>

A simple extraction protocol was developed to test the capability of NADES to solubilize curcuminoids from plant matrix. The powder of *C. zedoaria* was mixed with NADES in a bottle with cap (powder/NADES ratio = 20 mg powder/3 mL NADES) and stirred (350 rpm) at 40°C for 24 h. Triplicate samples of the resulting solution were diluted with water and analyzed with HPLC-DAD at a wavelength of 421.4 nm. The NADESs were prepared according to Dai et al. [20] with slight modification, i.e. by using freeze-drying instead of vacuum evaporation. The liquefied solid mixture can be called as NADES, when after the freeze-drying process it remains liquid and is visually clear and transparent with no precipitation and crystallization that are formed. It can be kept until a year without any changes in appearances and physical properties, i.e. density and viscosity. In addition, the purity of the individual component of NADES does not affect the

O and FG-H<sup>2</sup>

O, CCG-H<sup>2</sup>

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

O are neutral type and CAS-

O prepared with different

O, a yellowish color will be

O, although sucrose hydrolysis

O are the

29

H-<sup>1</sup> H-

O and CCF-H<sup>2</sup>

http://dx.doi.org/10.5772/intechopen.71755

O and CCMA-H<sup>2</sup>

NADES properties. As shown in **Figure 7**, the FT-IR spectra of CAS-H<sup>2</sup>

molecular structure of NADES [19, 20].

density nor viscosity of CAS-H<sup>2</sup>

of both CAS-H<sup>2</sup>

In the case of acidic type of NADESs, e.g. CAS-H<sup>2</sup>

O and MAS-H<sup>2</sup>

grades (purity) of citric acid are similar indicating that there are no structural changes.

In analogy with DES, the two components of NADES are particularly bonded by hydrogen bond [19]. The cross-correlation between sucrose and malic acid was observed by <sup>1</sup>

nuclear Overhauser enhancement spectroscopy (NOESY). It revealed molecular interactions of protons on the C2 and C3 positions of malic acid with those on the C1 and C2' of sucrose [19]. This analysis also suggests that water might also participate in the formation of super-

observed right after the extraction process takes place. It will be deepened along the elapsed extraction time due to the caramelization of sucrose promoted by the presence of acid. The pH

melization reaction is the hydrolysis of sucrose by protonation of the glycosidic linkage [42]. Though the extraction process was only heated at 40°C, the effect of acidic condition is comparable with the effect of heating at high temperature at neutral pH [43]. However, neither the

reaction (**Figure 8**). Nevertheless, sugar hydrolysis was not observed in other NADESs that

O and MAS-H<sup>2</sup>

also occurs at very concentrated sucrose solution even at neutral pH [42].

consist of sucrose such as in the neutral NADES type, FS-H<sup>2</sup>

O and MAS-H<sup>2</sup>

O is two (measured at 10x dilution with Aquadest). The cara-

O was significantly affected by the hydrolysis

O for the acidic type, whereas CCGo-H<sup>2</sup>

basic type. Meanwhile, the amphoteric types were excluded in this study.

represented by CCCA-H<sup>2</sup>

O and MAS-H<sup>2</sup>

H2

Traditionally curcumin has been used as food coloring, flavoring and preservative. Because of its wide spectrum of biological activity, an extensive number of studies have been focused on curcumin. Recently, curcumin has also been shown to display antioxidant, anticancer, antiviral, anti-infectious and anti-amyloidogenic properties. Numerous methods to isolate curcumin as well as other curcuminoids from *C. longa* rhizomes have been reported, such as conventional solvent extraction, hot and cold percolation, the use of alkaline solution and insoluble salt, supercritical carbon dioxide extraction, microwave-assisted extraction and ultrasonic-assisted extraction techniques. However, only a few of them use either green solvent or green process such as pressurized hot water extraction [35]. In the contrary, volatile organic solvents, e.g. methanol, ethanol, acetone and hexane, are still widely applied.

The capabilities of NADES to extract and stabilize bioactive compounds have been investigated by several authors. The solubilities of secondary metabolites such as rutin, quercetin, cinnamic acid, carthamin, taxol and ginkgolide B in different types of NADES have been studied by Choi et al. [19] and Dai et al. [20], while the stabilization ability of NADES on unstable natural colorants, carthamin and anthocyanins, during heating, storage and exposure to light has been reported by Dai et al. [36]. In addition, Bajkacz and Adamek et al. [37] also showed that NADES can be applied for isoflavone extraction. NADES can also be applied for protein stabilization [38], bioavailability improvement [39], antimicrobial agent [40] and bioactivity enhancement of plant extract [41]. Most of them used choline chloride-based NADES, the best combination of NADES which is suitable with their studied compounds. The broad range utilization of NADES showed that NADES leads a novel application in food and pharmaceutical industry.

The selection of solvent for extraction, i.e. liquid-liquid extraction (LLE), depends on its physical properties such viscosity, density and miscibility. It is convenient to select solvent with low viscosity to facilitate mixing as well as maximizing solvent penetration to the plant matrix but with a large density difference for the separation process. The inherent viscous properties of NADESs differ enormously according to their composition, but in all cases, it can be reduced by the addition of a certain amount of water. It should be noted that the addition of water changes the properties of NADESs, i.e. polarity, density and solubilizing and stabilizing capability. However, excessive dilution of NADESs, ca. approximately >50% weight of water, disrupts the special structure of NADESs due to the loss of the existing hydrogen bonds [20]. The viscosities of NADESs can also be decreased by increasing temperature. Generally, NADESs composed of sugar are the most viscous, while choline chloride-based NADESs are less viscous, while the glycerol-based NADESs are the least. Common efforts to minimize the resistance of viscosity and improving the extraction rate, such as mechanical agitation, microwave and ultrasound-assisted extraction, can be used with NADES.

To figure out the broad application of NADES in natural product extraction, this chapter documents the application of NADES on the extraction of curcumin, a low solubility phenolic compound in water, and galantamine, an alkaloid of acetylcholine inhibitor.

*Curcuma zedoaria* that contains 1.96% ± 0.07% of dry weight of curcuminoids was kindly donated by Sari Herbal (Sukun, Malang, Indonesia). It was chosen in this study since it is traditionally believed as an anticancer medicine in Indonesia. Twelve NADESs were used as solvents for extracting curcuminoids from fine powder of *C. zedoaria*, representing different types of NADESs: (1) ionic type, consisted of organic acids, i.e. citric acid, malic acid and lactic acid, and basic compounds such choline chloride or betaine; (2) neutral type, no ionic constituent, mixed of polyalcohol, i.e. glycerol, glycine, 1–2-propanediol and sugars; (3) acidic type, consisted of neutral compounds such sugars and acidic compounds; (4) basics type, consisted of basic compounds; and the last is (5) amphoteric type, consisted of combination of amino acids and sugars, polyalcohol or acidic compounds. In the case of ionic type of NADESs, it is represented by CCCA-H<sup>2</sup> O and CCMA-H<sup>2</sup> O; FS-H<sup>2</sup> O and FG-H<sup>2</sup> O are neutral type and CAS-H2 O and MAS-H<sup>2</sup> O for the acidic type, whereas CCGo-H<sup>2</sup> O, CCG-H<sup>2</sup> O and CCF-H<sup>2</sup> O are the basic type. Meanwhile, the amphoteric types were excluded in this study.

*Curcuma zedoaria* and *Curcuma manga*, a plant of the ginger family, with other two curcumi-

Traditionally curcumin has been used as food coloring, flavoring and preservative. Because of its wide spectrum of biological activity, an extensive number of studies have been focused on curcumin. Recently, curcumin has also been shown to display antioxidant, anticancer, antiviral, anti-infectious and anti-amyloidogenic properties. Numerous methods to isolate curcumin as well as other curcuminoids from *C. longa* rhizomes have been reported, such as conventional solvent extraction, hot and cold percolation, the use of alkaline solution and insoluble salt, supercritical carbon dioxide extraction, microwave-assisted extraction and ultrasonic-assisted extraction techniques. However, only a few of them use either green solvent or green process such as pressurized hot water extraction [35]. In the contrary, volatile

noid compounds: demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC).

organic solvents, e.g. methanol, ethanol, acetone and hexane, are still widely applied.

industry.

28 Green Chemistry

The capabilities of NADES to extract and stabilize bioactive compounds have been investigated by several authors. The solubilities of secondary metabolites such as rutin, quercetin, cinnamic acid, carthamin, taxol and ginkgolide B in different types of NADES have been studied by Choi et al. [19] and Dai et al. [20], while the stabilization ability of NADES on unstable natural colorants, carthamin and anthocyanins, during heating, storage and exposure to light has been reported by Dai et al. [36]. In addition, Bajkacz and Adamek et al. [37] also showed that NADES can be applied for isoflavone extraction. NADES can also be applied for protein stabilization [38], bioavailability improvement [39], antimicrobial agent [40] and bioactivity enhancement of plant extract [41]. Most of them used choline chloride-based NADES, the best combination of NADES which is suitable with their studied compounds. The broad range utilization of NADES showed that NADES leads a novel application in food and pharmaceutical

The selection of solvent for extraction, i.e. liquid-liquid extraction (LLE), depends on its physical properties such viscosity, density and miscibility. It is convenient to select solvent with low viscosity to facilitate mixing as well as maximizing solvent penetration to the plant matrix but with a large density difference for the separation process. The inherent viscous properties of NADESs differ enormously according to their composition, but in all cases, it can be reduced by the addition of a certain amount of water. It should be noted that the addition of water changes the properties of NADESs, i.e. polarity, density and solubilizing and stabilizing capability. However, excessive dilution of NADESs, ca. approximately >50% weight of water, disrupts the special structure of NADESs due to the loss of the existing hydrogen bonds [20]. The viscosities of NADESs can also be decreased by increasing temperature. Generally, NADESs composed of sugar are the most viscous, while choline chloride-based NADESs are less viscous, while the glycerol-based NADESs are the least. Common efforts to minimize the resistance of viscosity and improving the extraction rate, such as mechanical agitation, micro-

To figure out the broad application of NADES in natural product extraction, this chapter documents the application of NADES on the extraction of curcumin, a low solubility phenolic

wave and ultrasound-assisted extraction, can be used with NADES.

compound in water, and galantamine, an alkaloid of acetylcholine inhibitor.

A simple extraction protocol was developed to test the capability of NADES to solubilize curcuminoids from plant matrix. The powder of *C. zedoaria* was mixed with NADES in a bottle with cap (powder/NADES ratio = 20 mg powder/3 mL NADES) and stirred (350 rpm) at 40°C for 24 h. Triplicate samples of the resulting solution were diluted with water and analyzed with HPLC-DAD at a wavelength of 421.4 nm. The NADESs were prepared according to Dai et al. [20] with slight modification, i.e. by using freeze-drying instead of vacuum evaporation. The liquefied solid mixture can be called as NADES, when after the freeze-drying process it remains liquid and is visually clear and transparent with no precipitation and crystallization that are formed. It can be kept until a year without any changes in appearances and physical properties, i.e. density and viscosity. In addition, the purity of the individual component of NADES does not affect the NADES properties. As shown in **Figure 7**, the FT-IR spectra of CAS-H<sup>2</sup> O prepared with different grades (purity) of citric acid are similar indicating that there are no structural changes.

In analogy with DES, the two components of NADES are particularly bonded by hydrogen bond [19]. The cross-correlation between sucrose and malic acid was observed by <sup>1</sup> H-<sup>1</sup> Hnuclear Overhauser enhancement spectroscopy (NOESY). It revealed molecular interactions of protons on the C2 and C3 positions of malic acid with those on the C1 and C2' of sucrose [19]. This analysis also suggests that water might also participate in the formation of supermolecular structure of NADES [19, 20].

In the case of acidic type of NADESs, e.g. CAS-H<sup>2</sup> O and MAS-H<sup>2</sup> O, a yellowish color will be observed right after the extraction process takes place. It will be deepened along the elapsed extraction time due to the caramelization of sucrose promoted by the presence of acid. The pH of both CAS-H<sup>2</sup> O and MAS-H<sup>2</sup> O is two (measured at 10x dilution with Aquadest). The caramelization reaction is the hydrolysis of sucrose by protonation of the glycosidic linkage [42]. Though the extraction process was only heated at 40°C, the effect of acidic condition is comparable with the effect of heating at high temperature at neutral pH [43]. However, neither the density nor viscosity of CAS-H<sup>2</sup> O and MAS-H<sup>2</sup> O was significantly affected by the hydrolysis reaction (**Figure 8**). Nevertheless, sugar hydrolysis was not observed in other NADESs that consist of sucrose such as in the neutral NADES type, FS-H<sup>2</sup> O, although sucrose hydrolysis also occurs at very concentrated sucrose solution even at neutral pH [42].

**Figure 7.** FT-IR spectra of CAS-H<sup>2</sup> O, (A) using technical grade and (B) using pure grade of citric acid.

The resulted data of curcuminoid extraction with NADESs are shown in **Figure 9**. It is surprising that in overall NADESs show better extracting capability of curcuminoids than organic solvent, i.e. ethanol, and water although NADESs themselves are water-based solvents (**Figure 9**). Curcuminoids can be extracted by NADES due to the hydrogen bond formed between curcuminoids with NADES suggesting the presence of hydroxyl groups in curcumin, demethoxycurcumin as well as bisdemethoxycurcumin. At the same time, none of curcuminoids was extracted with both ethanol and water using the same extraction protocol as NADES. Curcumin is practically insoluble in water, i.e. ca. 4 ppb (4 μg/L at pH = 7,3) [44]. Curcuminoids were the best extracted by CCMA-H<sup>2</sup> O (1:1:3), 0.355 ± 0.019 mg/g, which is in agreement with that reported by Euterpio et al. [35] and Kwon and Chung [45]. A 0.136 mg curcuminoids/g of *C. longa* was obtained using a subcritical mixture of MeOH-H<sup>2</sup> O (50:50, v/v) at 135°C, 5 atm for 5 min [45], while pressurized hot water extraction (PHWE) yielded 0.503 and 0.204 mg curcuminoids/g *C. longa* at 90 and 250°C, respectively [35] although *C. longa* contains higher curcuminoids, i.e. ca. 4.4% of dry weight, than *C. zedoaria*.

polarity, water content or pH [40]. However, exhaustive extraction of *C. zedoaria* using Soxhlet and maceration with ethanol (96%) as solvent only gave 0.119 ± 0.0001 and 0.152 ± 0.010 mg curcuminoids/g dry weight, respectively. Degradation of curcumin might occur at high temperature (78°C) during Soxhlet extraction. Salem et al. [46] reported that curcumin degraded

O and (B) yellowish CAS-H<sup>2</sup>

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

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31

yield of 0.233 mg/g ± 0.017, i.e. approximately 36% less than that obtained after 24 h extraction. Though curcumin is reported to be stable at 10–55°C [46], prolonged exposure at 40°C

cumin, and extraction time may affect the obtained yield. In fact curcumin is precipitated

only 37–52% curcuminoids were left after 96 h. This coincides with Tonnesen and Karlsen [47] who reported that the degradation of curcumin will be faster approximately 100x than that in concentrated solution at pH < 7. It clearly concludes that water content may affect the stabilizing

after about 3 days due to its low water solubility at pH = 1–7 [47]. The CCMA-H<sup>2</sup>

had a pH of 2 at 10x dilution with Aquadest. The native pH of CCMA-H<sup>2</sup>

O (1:1:2) up to 96 h (4 days) only gave a

O.

O (2:1:26) (water content = 40% by weight),

O (1:1:2) could not stabilize cur-

O (1:1:2)

O (1:1:2) cannot be

after 24 h exposure at 70°C.

Longer curcuminoid extraction with CCMA-H<sup>2</sup>

**Figure 8.** FT-IR spectra: (A) clear and transparent CAS-H<sup>2</sup>

measured due to its inherent high viscosity.

In NADES with higher water content such as FS-H<sup>2</sup>

(ca. 96 h) could degrade curcumin. In addition, CCMA-H<sup>2</sup>

The neutral, ionic and basic types of NADESs give more or less similar yields of curcuminoids, while the lowest yields were obtained by acidic types of NADESs, i.e. CAS-H<sup>2</sup> O (1:2:15) and MAS-H<sup>2</sup> O (1:1:11) with yields of 0.151 ± 0.001 and 0.131 ± 0.002 mg/g, respectively. Though CCMA-H<sup>2</sup> O (1:1:2) yielded the highest curcuminoids, apparently there is no direct relation between solubilizing capacity of the NADES with respect to curcumin and the Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids http://dx.doi.org/10.5772/intechopen.71755 31

**Figure 8.** FT-IR spectra: (A) clear and transparent CAS-H<sup>2</sup> O and (B) yellowish CAS-H<sup>2</sup> O.

The resulted data of curcuminoid extraction with NADESs are shown in **Figure 9**. It is surprising that in overall NADESs show better extracting capability of curcuminoids than organic solvent, i.e. ethanol, and water although NADESs themselves are water-based solvents (**Figure 9**). Curcuminoids can be extracted by NADES due to the hydrogen bond formed between curcuminoids with NADES suggesting the presence of hydroxyl groups in curcumin, demethoxycurcumin as well as bisdemethoxycurcumin. At the same time, none of curcuminoids was extracted with both ethanol and water using the same extraction protocol as NADES. Curcumin is practically insoluble in water, i.e. ca. 4 ppb (4 μg/L at pH = 7,3) [44]. Curcuminoids were the best

O, (A) using technical grade and (B) using pure grade of citric acid.

by Euterpio et al. [35] and Kwon and Chung [45]. A 0.136 mg curcuminoids/g of *C. longa* was

while pressurized hot water extraction (PHWE) yielded 0.503 and 0.204 mg curcuminoids/g *C. longa* at 90 and 250°C, respectively [35] although *C. longa* contains higher curcuminoids, i.e. ca.

The neutral, ionic and basic types of NADESs give more or less similar yields of curcuminoids, while the lowest yields were obtained by acidic types of NADESs, i.e. CAS-H<sup>2</sup>

direct relation between solubilizing capacity of the NADES with respect to curcumin and the

O (1:1:3), 0.355 ± 0.019 mg/g, which is in agreement with that reported

O (1:1:11) with yields of 0.151 ± 0.001 and 0.131 ± 0.002 mg/g, respec-

O (1:1:2) yielded the highest curcuminoids, apparently there is no

O (50:50, v/v) at 135°C, 5 atm for 5 min [45],

O

extracted by CCMA-H<sup>2</sup>

**Figure 7.** FT-IR spectra of CAS-H<sup>2</sup>

30 Green Chemistry

(1:2:15) and MAS-H<sup>2</sup>

tively. Though CCMA-H<sup>2</sup>

4.4% of dry weight, than *C. zedoaria*.

obtained using a subcritical mixture of MeOH-H<sup>2</sup>

polarity, water content or pH [40]. However, exhaustive extraction of *C. zedoaria* using Soxhlet and maceration with ethanol (96%) as solvent only gave 0.119 ± 0.0001 and 0.152 ± 0.010 mg curcuminoids/g dry weight, respectively. Degradation of curcumin might occur at high temperature (78°C) during Soxhlet extraction. Salem et al. [46] reported that curcumin degraded after 24 h exposure at 70°C.

Longer curcuminoid extraction with CCMA-H<sup>2</sup> O (1:1:2) up to 96 h (4 days) only gave a yield of 0.233 mg/g ± 0.017, i.e. approximately 36% less than that obtained after 24 h extraction. Though curcumin is reported to be stable at 10–55°C [46], prolonged exposure at 40°C (ca. 96 h) could degrade curcumin. In addition, CCMA-H<sup>2</sup> O (1:1:2) could not stabilize curcumin, and extraction time may affect the obtained yield. In fact curcumin is precipitated after about 3 days due to its low water solubility at pH = 1–7 [47]. The CCMA-H<sup>2</sup> O (1:1:2) had a pH of 2 at 10x dilution with Aquadest. The native pH of CCMA-H<sup>2</sup> O (1:1:2) cannot be measured due to its inherent high viscosity.

In NADES with higher water content such as FS-H<sup>2</sup> O (2:1:26) (water content = 40% by weight), only 37–52% curcuminoids were left after 96 h. This coincides with Tonnesen and Karlsen [47] who reported that the degradation of curcumin will be faster approximately 100x than that in concentrated solution at pH < 7. It clearly concludes that water content may affect the stabilizing

**Figure 9.** Curcuminoids extracted (mg curcuminoids/g dry weight) from *Curcuma zedoaria* with different types of NADES (G = glucose, F = fructose, S = sucrose, CC = choline chloride, MA = malic acid, CA = citric acid and Go = glycerol).

ability of NADES to curcuminoids. Moreover, the low level of curcuminoids might also be due to the loss of NADES structure since the hydrogen bond will rupture when NADES is further diluted with water content higher than 50% [20]. Though higher water content minimized the mass transfer resistance between NADES and plant matrix, water dilution affects considerably the target compounds and the nature of NADES itself.

A profile of extracted curcuminoids was also studied to find the selectivity of NADES to curcuminoids (**Table 4**). It is shown that NADES and FS-H<sup>2</sup> O (2:1:26) are less selective to bisdemethoxyxurxumin (BDMC), but it is more selective to curcumin (ca. 54 wt.%) than to DMC (ca. 32 wt.%). Other kinds of NADESs also give different selectivity. The exhaustive extraction with 96% ethanol gave a similar selectivity, i.e. approximately 17, 23 and 60 wt.% for BDMC,


DMC and C, respectively. Hence, curcuminoid selectivity depends on the types of solvent. Moreover, in the case of curcuminoid extraction from *C. zedoaria* powder, the ratio of solid to NADES also affected the yield (data not shown). In conclusion, NADES is a better solvent to solubilize curcuminoids than water and ethanol. NADES is more selective to curcumin fol-

**Table 5.** Preliminary extraction of pressurized extraction for finding the best NADES extraction conditions.

Plant material Pwd = powder, Frz = freeze-dried. All the materials have 25–53 μm of particle size. Plant material and

Heat up and discharge time is in default setting. They were 1 and 5 min, respectively. All the experiments were

<sup>d</sup>Plant material, NADES and 1 g of sea sand were mixed prior loaded to the extractor cells. There were flushing with

Plant material, NADES and 1 g of sea sand were mixed prior loaded to the extractor cells. No flush with solvent (only with gas 1 min) between cycles. Around 3 g of sea sand was placed in the upper and lower part of the extractor cells.

Plant material, NADES and 1,5 g of sea sand were mixed prior loaded to the extractor cells. No flush with solvent (only with gas 1 min) between cycles. Around 2.5 g of sea sand was placed in the upper and lower part of the extractor cells. Abbreviations: β-alanine (**βA**), citric acid (**CA**), choline chloride (**CH**), glucose (**G**), malic acid (**MA**), L-Proline (**LPr**),

) between cycles, 1 min each. Around 3 g of sea sand was placed in the upper and lower part

**Trial Pressurized extraction conditions NADESc Results**

**Holding (min)b**

E1 <sup>d</sup> Pwd 100 30 15 50 100 CHCA (1:1) GMA

E3 <sup>d</sup> Pwd 300 60 2 50 50 MAS (1:1) CAS

E4 <sup>d</sup> Pwd 200 60 2 50 50 CHCA (1:1) βAMA

E5 <sup>e</sup> Pwd 200 60 2 50 50 CHCA (1:1) GMA

E6 <sup>f</sup> Pwd 200 60 2 50 50 MAS (1:1) CAS

**Temp (°C)**

**Press (bar)**

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

(1:1) LPrS (2:1)

http://dx.doi.org/10.5772/intechopen.71755

(1:1) GMA (1:1)

(1:1) LPrS (2:1)

(1:1) LPrS (2:1)

(1:1) βAMA (1:1)

Clogging

33

Clogging

Clogging

Clogging

None

**Preheat (min)**

The extraction of an alkaloid, galantamine, with NADES from its plant matrix *Narcissus pseudonarcissus sp. was* also conducted. A preliminary and simple extraction method similar to that of *C. zedoaria* explained above was also conducted with ground powder of *N. pseudonarcissus* with particle size around 25–53 μm. However, none of the galantamine was extracted although the extraction time was prolonged up to a week. This is probably because the mechanical stirring could not overcome the mass transfer resistance around the pellet of *N. pseudonarcissus* due to

lowed by DMC, while ethanol only gave 60% selectivity to curcumin.

the structure of plant matrix.

**Plant materiala**

a

b

c

e

f

sucrose (**S**).

conducted with two cycles.

solvent (water) and gas (N<sup>2</sup>

Ratio is in molar.

of the extractor cells.

**Weight (mg)**

NADES were mixed prior loaded to the extractor cells.

E2 <sup>d</sup> Frz 100 30 15 50 100

a Yield expressed with mg bioactive compound/g dry weight of *C. zedoaria.*

b Percentage of weight to the total weight of extracted *curcuminoids* (c*urcuminoid* = BDMC + DMC + C). c *Bisdemethoxycurcumin* (BDMC), *demethoxycurcumin* (DMC) and *curcumin* (C).

**Table 4.** Profile of the extracted *curcuminoids* (mg/g) with different extraction methods.


a Plant material Pwd = powder, Frz = freeze-dried. All the materials have 25–53 μm of particle size. Plant material and NADES were mixed prior loaded to the extractor cells.

b Heat up and discharge time is in default setting. They were 1 and 5 min, respectively. All the experiments were conducted with two cycles.

c Ratio is in molar.

**Extraction method Yield (mg/g a**

FS-H<sup>2</sup>

a

b

c

CCMA-H<sup>2</sup>

32 Green Chemistry

**) (% b) Extracted curcuminoids** 

O (2:1:26) are less selective to bis-

**(mg/g <sup>±</sup> SD) BDMCc DMC <sup>C</sup>**

ability of NADES to curcuminoids. Moreover, the low level of curcuminoids might also be due to the loss of NADES structure since the hydrogen bond will rupture when NADES is further diluted with water content higher than 50% [20]. Though higher water content minimized the mass transfer resistance between NADES and plant matrix, water dilution affects considerably

**Figure 9.** Curcuminoids extracted (mg curcuminoids/g dry weight) from *Curcuma zedoaria* with different types of NADES (G = glucose, F = fructose, S = sucrose, CC = choline chloride, MA = malic acid, CA = citric acid and Go = glycerol).

A profile of extracted curcuminoids was also studied to find the selectivity of NADES to

demethoxyxurxumin (BDMC), but it is more selective to curcumin (ca. 54 wt.%) than to DMC (ca. 32 wt.%). Other kinds of NADESs also give different selectivity. The exhaustive extraction with 96% ethanol gave a similar selectivity, i.e. approximately 17, 23 and 60 wt.% for BDMC,

Soxhlet (EtOH 96%) 0.020 (17) 0.028 (23) 0.071 (60) 0.120 ± 0.020 Maceration (EtOH 96%) 0.026 (18) 0.034 (21) 0.092 (61) 0.150 ± 0.660

Percentage of weight to the total weight of extracted *curcuminoids* (c*urcuminoid* = BDMC + DMC + C).

Yield expressed with mg bioactive compound/g dry weight of *C. zedoaria.*

the target compounds and the nature of NADES itself.

curcuminoids (**Table 4**). It is shown that NADES and FS-H<sup>2</sup>

*Bisdemethoxycurcumin* (BDMC), *demethoxycurcumin* (DMC) and *curcumin* (C).

**Table 4.** Profile of the extracted *curcuminoids* (mg/g) with different extraction methods.

O (2:1:26) 0.013 (14) 0.030 (32) 0.051 (54) 0.093 ± 0.002

O (1:1:2) 0.017 (5) 0.072 (20) 0.266 (75) 0.355 ± 0.019

<sup>d</sup>Plant material, NADES and 1 g of sea sand were mixed prior loaded to the extractor cells. There were flushing with solvent (water) and gas (N<sup>2</sup> ) between cycles, 1 min each. Around 3 g of sea sand was placed in the upper and lower part of the extractor cells.

e Plant material, NADES and 1 g of sea sand were mixed prior loaded to the extractor cells. No flush with solvent (only with gas 1 min) between cycles. Around 3 g of sea sand was placed in the upper and lower part of the extractor cells.

f Plant material, NADES and 1,5 g of sea sand were mixed prior loaded to the extractor cells. No flush with solvent (only with gas 1 min) between cycles. Around 2.5 g of sea sand was placed in the upper and lower part of the extractor cells. Abbreviations: β-alanine (**βA**), citric acid (**CA**), choline chloride (**CH**), glucose (**G**), malic acid (**MA**), L-Proline (**LPr**), sucrose (**S**).

**Table 5.** Preliminary extraction of pressurized extraction for finding the best NADES extraction conditions.

DMC and C, respectively. Hence, curcuminoid selectivity depends on the types of solvent. Moreover, in the case of curcuminoid extraction from *C. zedoaria* powder, the ratio of solid to NADES also affected the yield (data not shown). In conclusion, NADES is a better solvent to solubilize curcuminoids than water and ethanol. NADES is more selective to curcumin followed by DMC, while ethanol only gave 60% selectivity to curcumin.

The extraction of an alkaloid, galantamine, with NADES from its plant matrix *Narcissus pseudonarcissus sp. was* also conducted. A preliminary and simple extraction method similar to that of *C. zedoaria* explained above was also conducted with ground powder of *N. pseudonarcissus* with particle size around 25–53 μm. However, none of the galantamine was extracted although the extraction time was prolonged up to a week. This is probably because the mechanical stirring could not overcome the mass transfer resistance around the pellet of *N. pseudonarcissus* due to the structure of plant matrix.

Therefore, a pressurized extraction method using a pressurized extractor apparatus E-916 (Büchi, Flawil, Switzerland) was performed. It is a fast, simple and reproducible method facilitated by high-pressure condition instead of mechanical agitation. In addition, it is also a fast screening method to find the best extraction condition for NADES extraction. Preliminary experiments were conducted to find the best extraction configuration, whilst NADESs are used as a solvent as shown in **Table 5**.

organic solvents. Besides that, the use of DESs could lead to a simpler extraction or separation procedure, especially in a large industrial scale, due to non-toxic and inflammable nature of DES. However, at the same time, one has to consider that there is no universal solvent for all kinds of compounds. Each material and targeted compound requires the development of a specific process. No single standard procedure of extraction is suitable for extractions of all secondary metabolites. This also allows a rather selective extraction which is particularly of interest for the isolation of pure compounds. Some parameters that should be considered in the development of a DES-extraction procedure are types of the matrix plant, type of extraction process, ratio of plant material to solvent and extraction conditions: temperature, duration of extraction,

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

35

, Adi Tjipto Utomo1

1 Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS

[1] Abidin MHZ, Hayyan M, Hayyan A, Jayakumar NS. New horizons in the extraction of bioactive compounds using deep eutectic solvents: A review. Analytica Chimica Acta.

[2] Vieira FA, Guilherme RJR, Neves MC, Abreu H, Rodrigues ERO, Maraschin M, Coutinho JAP, Ventura SPM. Single-step extraction of carotenoids from brown macroalgae using

[3] Wang L, Weller CL. Recent advances in extraction of nutraceuticals from plants. Trends

[4] Deetlefs M, Seddon KR. Assessing the greenness of some typical laboratory ionic liquid

[5] Zhang Q, Vigier KDO, Royer S, Jerome F. Deep eutectic solvents: Syntheses, properties

[6] Zhang S, Ye L, Zhang H, Hou J. Green-solvent-processable organic solar cells. Materials

nonionic surfactants. Separation and Purification Technology. 2017;**172**:268-276

2 Department of Chemical Engineering, Mulawarman University, Samarinda, Indonesia

3 Department of Chemical Engineering, National Taiwan University of Science and

, Helda Niawanti<sup>2</sup>

and Yi Hsu Ju<sup>3</sup>

viscosity and water content of DES.

Keputih Sukolilo, Surabaya, Indonesia

Technology, Taipei, Taiwan

2017;**979**:1-23

Today. 2016;**19**:533-543

\*, Orchidea Rachmaniah1

\*Address all correspondence to: szulle@chem-eng.its.ac.id

in Food Science and Technology. 2006;**17**:300-312

and applications. Chemical Society Reviews. 2012;**41**:7108-7146

preparations. Green Chemistry. 2010;**12**:17-30

**Author details**

Siti Zullaikah<sup>1</sup>

**References**

Two different kinds of plant matrix were used, i.e. ground powder and freeze-dried powder of *N. pseudonarcissus* bulb. Both have particle sizes of 25–53 μm. NADES with mild viscosity was chosen to minimize clogging problem inside the extraction cell which is essentially a packed bed where the bulk porosity is important to make a good contact between NADES and plant matrix. If the sample inside the extraction cell is too compact, it creates a clogging problem and overpressurized the cell; otherwise the contact between the solute and solvent will be minimal leading to a low yield.

At high-pressure conditions (100 bar), E1 and E2, NADESs were overcooked, and the sugar was caramelized although only 30 min of preheating time was applied. Therefore, the extractions were performed at a lower pressure (50 bar). To compensate the pressure reduction, the preheating time was increased. Finally, extraction condition E6 was found to be free of clogging and overcooked problems. Further extraction of galantamine with NADES by applying E6 condition gave 6.11 and 9.33 mg of galantamine/g dry weight for CAS (1:1) and MAS (1:1), respectively. These yields were higher than that obtained by water extraction as a control (5.35 mg of galantamine/g dry weight). These values were also higher than galantamine extraction with supercritical CO2 , i.e. 303 μg/g dry weight (70°C, 220 bar, 3 h) [48]. The selectivity of NADES to galantamine was slightly better (70–78%) than that of SC-CO<sup>2</sup> (<70%). Thus, NADES extraction of galantamine is more efficient, in terms of both galantamine yield and selectivity.

#### **5. Concluding remarks**

Deep eutectic solvents (DESs), including natural deep eutectic solvents (NADESs), are new generation of solvents. DES can be prepared by mixing two or more components at eutectic composition so that the mixture has lower melting point than those of the constituent components. DESs are typically prepared from quaternary ammonium halide salts and hydrogen bond donors (HBDs), such as amide, carboxylic acid, alcohol, sugar, amino or organic acid and alkylsulfate or alkyl phosphate. The behaviors and properties of DESs can be adjusted by varying the hydrogen bond donors and their molar ratio in the mixtures [13]. The polarities of DESs can match those of conventional organic solvents; however, DESs have several advantages, such as non-toxic, non-volatile, inflammable and biodegradable. Therefore, DESs can be considered as green solvents.

DESs potentially have unlimited number of applications. The applications of DESs to purify crude biodiesel made from rice bran oil and to extract natural bioactive compounds, such as oryzanol, cucurmin and galantamine, have been discussed in this chapter. DESs could give higher extraction yields of natural bioactive compounds than those obtained using conventional organic solvents. Besides that, the use of DESs could lead to a simpler extraction or separation procedure, especially in a large industrial scale, due to non-toxic and inflammable nature of DES.

However, at the same time, one has to consider that there is no universal solvent for all kinds of compounds. Each material and targeted compound requires the development of a specific process. No single standard procedure of extraction is suitable for extractions of all secondary metabolites. This also allows a rather selective extraction which is particularly of interest for the isolation of pure compounds. Some parameters that should be considered in the development of a DES-extraction procedure are types of the matrix plant, type of extraction process, ratio of plant material to solvent and extraction conditions: temperature, duration of extraction, viscosity and water content of DES.

## **Author details**

Therefore, a pressurized extraction method using a pressurized extractor apparatus E-916 (Büchi, Flawil, Switzerland) was performed. It is a fast, simple and reproducible method facilitated by high-pressure condition instead of mechanical agitation. In addition, it is also a fast screening method to find the best extraction condition for NADES extraction. Preliminary experiments were conducted to find the best extraction configuration, whilst NADESs are

Two different kinds of plant matrix were used, i.e. ground powder and freeze-dried powder of *N. pseudonarcissus* bulb. Both have particle sizes of 25–53 μm. NADES with mild viscosity was chosen to minimize clogging problem inside the extraction cell which is essentially a packed bed where the bulk porosity is important to make a good contact between NADES and plant matrix. If the sample inside the extraction cell is too compact, it creates a clogging problem and overpressurized the cell; otherwise the contact between the solute and solvent

At high-pressure conditions (100 bar), E1 and E2, NADESs were overcooked, and the sugar was caramelized although only 30 min of preheating time was applied. Therefore, the extractions were performed at a lower pressure (50 bar). To compensate the pressure reduction, the preheating time was increased. Finally, extraction condition E6 was found to be free of clogging and overcooked problems. Further extraction of galantamine with NADES by applying E6 condition gave 6.11 and 9.33 mg of galantamine/g dry weight for CAS (1:1) and MAS (1:1), respectively. These yields were higher than that obtained by water extraction as a control (5.35 mg of galantamine/g dry weight). These values were also higher than galantamine extrac-

extraction of galantamine is more efficient, in terms of both galantamine yield and selectivity.

Deep eutectic solvents (DESs), including natural deep eutectic solvents (NADESs), are new generation of solvents. DES can be prepared by mixing two or more components at eutectic composition so that the mixture has lower melting point than those of the constituent components. DESs are typically prepared from quaternary ammonium halide salts and hydrogen bond donors (HBDs), such as amide, carboxylic acid, alcohol, sugar, amino or organic acid and alkylsulfate or alkyl phosphate. The behaviors and properties of DESs can be adjusted by varying the hydrogen bond donors and their molar ratio in the mixtures [13]. The polarities of DESs can match those of conventional organic solvents; however, DESs have several advantages, such as non-toxic, non-volatile, inflammable and biodegradable. Therefore, DESs

DESs potentially have unlimited number of applications. The applications of DESs to purify crude biodiesel made from rice bran oil and to extract natural bioactive compounds, such as oryzanol, cucurmin and galantamine, have been discussed in this chapter. DESs could give higher extraction yields of natural bioactive compounds than those obtained using conventional

NADES to galantamine was slightly better (70–78%) than that of SC-CO<sup>2</sup>

, i.e. 303 μg/g dry weight (70°C, 220 bar, 3 h) [48]. The selectivity of

(<70%). Thus, NADES

used as a solvent as shown in **Table 5**.

34 Green Chemistry

will be minimal leading to a low yield.

tion with supercritical CO2

**5. Concluding remarks**

can be considered as green solvents.

Siti Zullaikah<sup>1</sup> \*, Orchidea Rachmaniah1 , Adi Tjipto Utomo1 , Helda Niawanti<sup>2</sup> and Yi Hsu Ju<sup>3</sup>

\*Address all correspondence to: szulle@chem-eng.its.ac.id

1 Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS Keputih Sukolilo, Surabaya, Indonesia

2 Department of Chemical Engineering, Mulawarman University, Samarinda, Indonesia

3 Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan

#### **References**


[7] Almeida MR, Passos H, Pereira MM, Lima AS, Coutinho JAP, Freire MG. Ionic liquids as additives to enhance the extraction of antioxidants in aqueous two-phase systems. Separation and Purification Technology. 2014;**128**:1-10

[22] Ju YH, Zullaikah S. Effect of acid-catalyzed methanolysis on the bioactive components of rice bran oil. Journal of the Taiwan Institute of Chemical Engineers. 2013;**44**:924-928

Green Separation of Bioactive Natural Products Using Liquefied Mixture of Solids

http://dx.doi.org/10.5772/intechopen.71755

37

[23] Zullaikah S, Lai CC, Vali SR, Ju YH. A two-step acid-catalyzed process for the production of biodiesel from rice bran oil. Bioresource Technology. 2005;**96**:1889-1896

[24] Lin L, Ying D, Chaitep S, Vittayapadung S. Biodiesel production from crude rice bran oil

[25] Shiu PJ, Gunawan S, Hsieh WH, Kasim NS, Ju YH. Biodiesel production from rice bran

[26] Zullaikah S, Rahkadima YT, Ju YH. A non-catalytic in situ process to produce biodiesel from a rice milling by-product using a subcritical water-methanol mixture. Renewable

[27] European Committee for Standardization (CEN). EN14214:2003. Automotive fuels–fatty acid methyl esters (FAME) for diesel engines—requirements and test methods. 2003

[28] Perkins SL, Painter P, Colina CM. Experimental and computational studies of choline chloride-based deep eutectic solvents. Journal of Chemical and Engineering Data.

[29] Wagle DV, Deakyne CA, Baker GA. Quantum chemical insight into the interactions and thermodynamics present in choline chloride based deep eutectic solvent. The Journal of

[30] Shahbaz K, Mjalli FS, Hashim MA, AlNashef IM. Using deep eutectic solvents for the removal of glycerol from palm oil-based biodiesel. Journal of Applied Sciences. 2010;**10**:

[31] Hayyan M, Aissaoui T, Hashim MA, AlSaadi MAH, Hayyan A. Triethylene glycol based deep eutectic solvents and their physical properties. Journal of the Taiwan Institute of

[32] Aissaoui T. Novel contribution to the chemical structure of choline chloride based deep

[33] Smith BC. Infrared spectral interpretation: A systematic approach. CRC Press United

[34] Niawanti H, Zullaikah S. Removal of bioactive compound (γ-Oryzanol) from Rice bran oil-based biodiesel using deep eutectic solvent. Chemical Engineering Transaction.

[35] Euterpio MA, Cavaliere C, Capriotti AL, Carlo C. Extending the applicability of pressurized hot water extraction to compounds exhibiting limited water solubility by pH control: Curcumin from the turmeric rhizome. Analytical and Bioanalytical Chemistry.

eutectic solvent. Pharmaceutica Analytica Acta. 2015;**6**:2153-2435

by a two-step in-situ process. Bioresource Technology. 2010;**101**:984-989

and properties as fuel. Applied Energy. 2009;**86**:681-688

Energy. 2017;**111**:764-770

2014;**59**:3652-3662

3349-3354

States. 1998

2017;**56**:1513-1518

2011;**401**:2977-2985

Physical Chemistry B. 2016;**120**:6739-6746

Chemical Engineers. 2015;**50**:24-30


[22] Ju YH, Zullaikah S. Effect of acid-catalyzed methanolysis on the bioactive components of rice bran oil. Journal of the Taiwan Institute of Chemical Engineers. 2013;**44**:924-928

[7] Almeida MR, Passos H, Pereira MM, Lima AS, Coutinho JAP, Freire MG. Ionic liquids as additives to enhance the extraction of antioxidants in aqueous two-phase systems.

[8] Gorke J, Srienc F, Kazlauskas R. Toward advanced ionic liquids. Polar, enzyme-friendly solvents for biocatalysis. Biotechnology and Bioprocess Engineering. 2010;**15**:40-53

[9] Wood N, Stephens G. Accelerating the discovery of biocompatible ionic liquids. Physical

[10] Domínguez de María P, Maugeri Z. Ionic liquids in biotransformations: From proof ofconcept to emerging deep-eutectic-solvents. Current Opinion in Chemical Biology. 2011;**15**:

[11] Pham TPT, Cho CW, Yun YS. Environmental fate and toxicity of ionic liquids: A review.

[12] Kareem MA, Mjalli FS, Hashim MA, Alnashef IM. Phosphonium-based ionic liquids analogues and their physical properties. Journal of Chemical & Engineering Data.

[13] Abbott AP, Boothby D, Capper G, Davies DL, Rasheed RK. Deep eutectic solvents formed between choline chloride and carboxylic acids: Versatile alternatives to ionic liq-

[14] Abbott AP, Capper G, Davies DL, Rasheed RK, Tambyrajah V. Novel solvent properties

[15] Smith EL, Abbot AP, Ryder KS. Deep eutectic solvent (DESs) and their applications.

[16] Gutiérrez MC, Ferrer ML, Mateo CR, del Monte F, Freeze-drying of aqueous solutions of deep eutectic solvents: A suitable approach to deep eutectic suspensions of self-assem-

[17] Imperato G, Eibler E, Niedermaier J, Konig B. Low-melting sugar-urea-salt mixtures as solvents for Diels-Alder reactions. Chemical Communications. 2005;**9**:1170-1172

[18] Francisco M, van den Bruinhorst A, Kroon MC. Low-transition-temperature mixtures (LTTMs): A new generation of designer solvents, Angewandte Chemie International

[19] Choi YH , van Spronsen J, Dai y, Verberne M, Hollmann F, Arends IWCE , Witkamp GJ, verpoorte R. Verpoorte, Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology? Plant Physiology. 2011;**156**:1701-1705 [20] Dai Y, van Spronsen J, Witkamp GJ , Verpoorte R . Choi YH. Natural deep eutectic solvents as a new potential media for green technology. Analytica Chimica Acta. 2013;**766**:61-68

[21] Ju YH, Vali SR. Rice bran oil as a potential resource for biodiesel: A review. Journal of

of choline chloride/urea mixtures. Chemical Communications. 2003;**1**:70-71

uids. Journal of the American Chemical Society. 2004;**126**:9142-9147

Separation and Purification Technology. 2014;**128**:1-10

Chemistry Chemical Physics. 2010;**12**:1670-1674

Water Research. 2010;**44**:352-372

Chemical Reviews. 2014;**114**:11060-11082

bled structures, Langmuir. 2009;**25**:5509-5515

Scientific and Industrial Research. 2005;**64**:866-882

Edition. 2013;**52**:3074-3085

220-225

36 Green Chemistry

2010;**55**:4632-4637


[36] Dai Y, Verpoorte R., Choi YH. Natural deep eutectic solvents providing enhanced stability of natural colorants from safflower (Carthamus tinctorius). Food Chemistry. 2014;**159**:116-121

**Chapter 3**

**Provisional chapter**

**Solventless Extraction of Essential Oil**

**Solventless Extraction of Essential Oil**

DOI: 10.5772/intechopen.72401

Essential oil is one of an important concentrated liquid that possesses many physical, chemical and pharmacological properties. Extraction of essential is one of the main issues in the last decade. Conventional treatment consisting of hydrodistillation and steam distillation has many disadvantages and finds difficult to purify essential oil. Now, it is much easier to extract essential oil with the invention of new greener technologies that reduce the involvement of solvent, decrease the extraction time, energy and descent the interaction of the concentrated volatile liquid with atmospheric oxygen through the

**Keywords:** solventless extraction, solvent-free extraction, essential oil, extraction

Essential oils are complex concentrated liquids comprise of volatile compounds. They have been extracted from numerous plant [1]. They have been widely used as a food preservative (eucalyptus essential oil, thyme), cosmetic preparation (lavender oil), antimicrobial (lemon grass, cumin, fennel), and anticancer agent (lemon grass, *Croton flavens*). Hydrodistillation and steam distillation is the common conventional method of extracting the essential oil [2–9]. These methods have some disadvantages Preservation of essential oil from its environment can be possible through a number of technologies such as nanospheres, liposome, microcapsules and nanoemulsions [10]. A lot of research is currently underway for extracting essential oil through new green methods. This review chapter documents the updated information

Muhammad Syarhabil Ahmad and Sarwat Ali Raja

© 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,

© 2018 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.

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

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Muhammad Shahzad Aslam,

Sarwat Ali Raja

**Abstract**

**1. Introduction**

Muhammad Shahzad Aslam,

Muhammad Syarhabil Ahmad and

http://dx.doi.org/10.5772/intechopen.72401

application of vacuum.

technology, green extraction

about novel solvent-less extraction of essential oil.


**Provisional chapter**

## **Solventless Extraction of Essential Oil**

**Solventless Extraction of Essential Oil**

Muhammad Shahzad Aslam,

[36] Dai Y, Verpoorte R., Choi YH. Natural deep eutectic solvents providing enhanced stability of natural colorants from safflower (Carthamus tinctorius). Food Chemistry.

[37] Bajkacz S, Adamek J. Evaluation of new natural deep eutectic solvents for the extraction

[38] Xin R, Qi S, Zeng C, Khan FI, Yang B, Wang Y. A functional natural deep eutectic solvent based on trehalose: Structural and physicochemical properties. Food Chemistry.

[39] Huang D, Zhang S, Zhong T, Ren W, Yao X, Guo Y, Duan XC, Yin YF, Zhang SS, Zhang X. Multi-targeting NGR-modified liposomes recognizing glioma tumor cells and vasculogenic mimicry for improving anti-glioma therapy. Oncotarget. 2016;**7**:43616-43628

[40] Wikene KO, Bruzell E, Tønnesen HH. Characterization and antimicrobial phototoxicity of curcumin dissolved in natural deep eutectic solvents. European Journal of Pharmaceutical

[41] Radošević K, Ćurko N, Srček VG, Bubalo MC, Tomašević M, Ganić KK, Redovniković IR. Natural deep eutectic solvents as beneficial extractants for enhancement of plant

[42] Clarke MA, Edye LA, Eggleston G. Sucrose decomposition in aqueous solution, and losses in sugar manufacture and refining. Advances in Carbohydrate Chemistry and

[43] Quintas M, Guimarães C, Baylina J, Brandão TRS, Silva CLM. Multiresponse modelling of the caramelisation reaction. Innovative Food Science & Emerging Technologies.

[44] Patra D, Sleem F. A new method for pH triggered curcumin release by applying poly (L-lysine) mediated nanoparticle-congregation. Analytica Chimica Acta. 2013;**795**:60-68

[45] Kwon HL, Chung MS. Pilot-scale subcritical solvent extraction of curcuminoids from cur-

[46] Salem M, Rohani S, Gillies ER. Curcumin, a promising anti-cancer therapeutic: A review of its chemical properties, bioactivity and approaches to cancer cell delivery. Royal

[47] Tønnesen HH, Karlsen J. Studies on curcumin and curcuminoids, Zeitschrift für Lebensmittel-

[48] Rachmaniah O, Choi YH, Arruabarrena I, Vermeulen B, Verpoorte R, van Spronsen J, Verpoorte R, Witkamp GJ. Environmentally benign super critical CO2 extraction of galantamine from floricultural crop waste of Narcissus Pseudonarcissus. The Journal of

extracts bioactivity. LWT-Food Science and Technology. 2016;**73**:45-51

of isoflavones from soy products. Talanta. 2017;**168**:329-335

2014;**159**:116-121

38 Green Chemistry

2017;**217**:560-567

Sciences. 2015;**80**:26-32

Biochemistry. 1997;**52**:441-470

cuma long L. Food Chemistry. 2015;**185**:58-64

Society of Chemistry Advance. 2014;**4**:10815-10829

Untersuchung und. Forschung. 1985;**180**:402-404

Supercritical Fluids. 2014;**93**:7-19

2007;**8**:306-315

Muhammad Shahzad Aslam, Muhammad Syarhabil Ahmad and Sarwat Ali Raja Muhammad Syarhabil Ahmad and Sarwat Ali Raja

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.72401

#### **Abstract**

Essential oil is one of an important concentrated liquid that possesses many physical, chemical and pharmacological properties. Extraction of essential is one of the main issues in the last decade. Conventional treatment consisting of hydrodistillation and steam distillation has many disadvantages and finds difficult to purify essential oil. Now, it is much easier to extract essential oil with the invention of new greener technologies that reduce the involvement of solvent, decrease the extraction time, energy and descent the interaction of the concentrated volatile liquid with atmospheric oxygen through the application of vacuum.

DOI: 10.5772/intechopen.72401

**Keywords:** solventless extraction, solvent-free extraction, essential oil, extraction technology, green extraction

#### **1. Introduction**

Essential oils are complex concentrated liquids comprise of volatile compounds. They have been extracted from numerous plant [1]. They have been widely used as a food preservative (eucalyptus essential oil, thyme), cosmetic preparation (lavender oil), antimicrobial (lemon grass, cumin, fennel), and anticancer agent (lemon grass, *Croton flavens*). Hydrodistillation and steam distillation is the common conventional method of extracting the essential oil [2–9]. These methods have some disadvantages Preservation of essential oil from its environment can be possible through a number of technologies such as nanospheres, liposome, microcapsules and nanoemulsions [10]. A lot of research is currently underway for extracting essential oil through new green methods. This review chapter documents the updated information about novel solvent-less extraction of essential oil.

© 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 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.

#### **2. Solventless extraction in a closed system**

Solventless extraction of essential oil was designed in a closed system under reduced pressure using a vacuum and compared the results with the conventional methods such as hydrodistillation. The quality and quantity of essential oil extracted from *Chromalaena odorata, Citronella*, *Baeckea frutescens*, Orange peel was found better than traditional method [11, 12]. The novel closed system was also optimized using central composite design (CCD) on *Chromalaena odorata* and maximum extraction yield was found to be under an ideal condition at 80°C temperature and 8 h of time [13].

**Medicinal plants**

*Cuminum cyminum* L. and *Zanthoxylum bungeanum* Maxim

*Rosmarinus officinalis* L.

*Dryopteris fragrans*

*Schisandra chinensis* (Turcz.) Baill

*Cajanus cajan* (L.) Millsp.

**Identified essential oil Pharmacological** 

2-Methyl-5-(1-methylethyl)-bicyclo[3.1.0]hex-2-ene, 1-Methylethylideneyl-cyclohexane, α-Pinene, Camphene, β-Phellandrene, β-Pinene, β-Myrcene, β-Phellandrene 3-Carene, 4-Carene, 1-Methyl-2-(1-methylethyl) benzene, d-Limonene, Eucalyptol, 1-Methyl-4-(1-methylethyl)-1,4-cyclohexadiene, 1-Methyl-4-(1-methylethylidene)-cyclohexene, β-Terpineol, 6,6-Dimethyl-2-methylene-bicyclo[2.2.1]heptan-3-one, 4-Methyl-1-(1-methylethyl)-3-cyclohexen-1-ol, Pulegone, Cuminal 4-(1-Methylethyl)-1-cyclohexene-1-carboxaldehyde 2-Ethylidene-6-methyl-3,5-heptadienal, α-Proyl-benzenemethanol,

4-(1-Methylethyl)-1,4-cyclohexadiene-1-methanol,

Caryophyllene oxide, Carotol

6-Isopropylidene-1-methyl-bicyclo[3.1.0]hexane, Caryophyllene, 2,6-Dimethyl-6-(4-methyl-3-pentenyl)-bicyclo[3.1.1]hept-2-ene, 7,11-Dimethyl-3-methylene-1,6,10-dodecatriene, 2-Isopropyl-5 methyl-9-methylene-bicyclo[4.4.0]dec-1-ene, Octahy-dro-3,8,8 trimethyl-6-methylene-1H-3a,7-methanoazulene, Thujopsene, 1-(1,5-Dimethyl-4-hexenyl)-4-methylbenzene 5-(1,5-Dimethy-4 hexenyl)-2-methyl-1,3-cyclohexadiene, Copaene, 1-Methyl-4-(5 methyl-1-methylene-4-hexenyl-cyclohexene, β-Sesquiphellandrene,

a-Pinene, Camphene, b-Pinene, Myrcene, a-Phellanderene, a-Terpinene c-Terpinene, Linalool, 1 1,8-Cineole, Camphor, Borneol, b-Caryophyllene, Trans b-ocimene, cis-Sabinene hydrate, Verbinone, Terpene-4-ol, Myrtenol, Bornyl acetate, Cis-jasmone, a-Humulene, Pentasiloxane Caryophyllene, 1,5-Diphenyl 2H-1,2,4 triazoline, 1-Methyl-2,4 nitrophenyl benzimid, 2-Methoxy-3,8-dioxocephalotax-1-ene, 1,2-Benzenedicarboxylic acid, 9-Octadecenoic acid, Docosanoic acid

Cedrene [1S-(1a,4a,7a)]-1,2,3,4,5,6,7,8-octahydro-1,4,9,9-tetramethyl-4,7-methanoazulene Caryophyllene, 10S,11S-himachala-3(12),4-diene, 4-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-2-butanone, (E)-4-(2,6,6 trimethyl-2-cyclohexen-1-yl)-3-buten-2-one, 1,2,3,4,4a,5,6,8a-Octahydro-7-methyl-4-methylene-1-(1-methylethyl)naphthalene, (R)-c-cadinene 4-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-3-buten-2-one, (1a,4ab,8aa)-1,2,3,4,4a,5,6,8a-Octahydro-7-methyl-4-methylene-1-(1 methylethyl)naphthalene, Albicanol, [1R-(1a,4ab,8aa)]-decahydro-1,4a-dimethyl-7-(1-methylethylidene)-1-naphthalenol, Calarene epoxide, (1R,4S,11R)-4,6,6,11-tetramethyltricyclo[5.4.0.0(4,8)] undecan-1-ol, 1,4,4a,5,6,7,8,8a-Octahydro-2,5,5,8a-tetramethyl-1 naphthalenemethanol, (-)-Isolongifolol, acetate, 1R,4S,7S,11R-2,2,4,8 tetramethyltricyclo[5.3.1.0(4,11)]undec-8-ene, [3S-(3a,5a,8a)]-

1,2,3,4,5,6,7,8-octahydro-a,a,3,8-tetramethyl-5-azulenemethanol acetate,

Cyclocopacamphene, Ylangene, beta-bourbonene, (+)-Sativene,(−)-beta-Elemene, Germacrene-D, (E)-(b)-Farnesene, (E)-a-bergamotene, Elixene, alpha-amorphene,(−)-beta-Chamigrene, Beta-bisabolene, b-maaliene, c-cadinene, b-himachalene, a-Chamigrene, (+)-a-Longipinene, (+)-Cuparene, b-Caryophyllene, Guaiene, (+)-Ledene, L-calamenene

3,6-Dimethyl-octane, Naphthalene, Dodecane, 6-Ethyl-undecane, 4-Methyl-dodecane, 4-Ethyl-undecane, 4,6-Dimethyl-dodecane, 1-Methyl-naphthalene, 2,6,11-Trimethyl-dodecane, α-Longipinene, 2-Methyl-tridecane, (+)-Cyclosativene, Ylangene, α-Copaene, Tetradecane, Longifolene, Caryophyllene, β-Selinene, α-Bergamotene, α-Himachalene, Humulene, Alloaromadendrene, α-Bisabolene, 2,4-Bis(1,1-dimethylethyl)-phenol, б-Cadinene, Hexadecane, Norphytane

a-Pinene, Camphene, 2-Carene, D-Limonene, o-Cymene, gamma-Terpinene, Thymol methyl ether, L-Bornyl acetate, **activities**

N.F [16]

Solventless Extraction of Essential Oil http://dx.doi.org/10.5772/intechopen.72401

Anti-bacterial [31]

Anti-oxidant [26]

Anti-oxidant [22]

Anti-microbial activities

[21]

**Reference**

41

#### **3. Solvent-free microwave extraction**

Solvent-free microwave extraction (SFME) of volatile natural substances was the first to patent in 2004. Farid Chemat et al. invented a method of extraction of essential oil consisting of a microwave oven with a microwave chamber for receiving the biological material and a condensation chamber. It was first tested on different spices such as ajowan (*Carum ajowan*, Apiaceae), cumin (*Cuminum cyminum*, Umbelliferae), star anise (*Illicium anisatum*, Illiciaceae) and the result had published on 4 February 2004. This technique is more fast, without solvent and effective when compared to hydrodistillation [14]. It was also evaluated on three aromatic herbs basil (*Ocimum basilicum*), garden mint (*Mentha crispa*), and thyme (*Thymus vulgaris*) where extraction time was significantly decreased from 4.5 h (hydro-distillation) to 30 min (SFME) [15]. This method was modified by many scientists. SFME method was modified by Wang et al. on dried *Cuminum cyminum* L. and *Zanthoxylum bungeanum* Maxim. by adding carbonyl iron powders (CIP) and mixed with the sample. CIP helps to reduce time (30 min) and microwave power (85 W) [16]. An attempt had been made to improve solvent-free extraction of essential oil using graphite powder, activated carbon powder. The effect was studied on *Illicium verum* Hook. f. and *Zingiber officinale* Rosc. [17]. Pressurized solvent-free microwave assisted extraction was used for extraction of phenolic compounds. The best extraction conditions were obtained, in a laboratory scale extractor of 50 mL filled with 4 g fresh berries, using a 1000 W microwave power applied during 50 s and repeated 5 cycles [18]. Solvent-free microwave extraction was modified by changing the flow of product toward the gravitation force. It is also known as Microwave dry-diffusion and gravity. It was better than hydrodistillation. The extraction performed in just 45 minutes with less energy and a clean process [19]. The extraction condition of SFME was optimized on *Elettaria cardamomum* (L.) using central composite design (CCD). The conditions such as time (min), power (W), humidity (%) was optimized by (CCD) and percentage yield (%) of was compared [20]. Optimization of SFME of pigeon pea leaves performed on an aliquot of 200 g plant materials that were wetted before extraction by soaking in water for 1 h [21]. Optimum parameters of SFME was performed on *S. chinensis* fruits and found ideal extraction time of 30 min, irradiation power 385 W and the moisture content of 68% respectively [22]. The quality of essential oil was also evaluated using Solvent-free microwave extraction method and compared with conventional method. It was found to be more effective than conventional method [23]. The effect of solvent-free microwave extraction was performed on several medicinal plants such as *Calamintha nepeta* [24], Basil leaves [25], *Dryopteris fragrans* [26], *Schisandra chinensis* [27]*, Cymbopogon winterianus* [28].


**2. Solventless extraction in a closed system**

**3. Solvent-free microwave extraction**

ture and 8 h of time [13].

40 Green Chemistry

Solventless extraction of essential oil was designed in a closed system under reduced pressure using a vacuum and compared the results with the conventional methods such as hydrodistillation. The quality and quantity of essential oil extracted from *Chromalaena odorata, Citronella*, *Baeckea frutescens*, Orange peel was found better than traditional method [11, 12]. The novel closed system was also optimized using central composite design (CCD) on *Chromalaena odorata* and maximum extraction yield was found to be under an ideal condition at 80°C tempera-

Solvent-free microwave extraction (SFME) of volatile natural substances was the first to patent in 2004. Farid Chemat et al. invented a method of extraction of essential oil consisting of a microwave oven with a microwave chamber for receiving the biological material and a condensation chamber. It was first tested on different spices such as ajowan (*Carum ajowan*, Apiaceae), cumin (*Cuminum cyminum*, Umbelliferae), star anise (*Illicium anisatum*, Illiciaceae) and the result had published on 4 February 2004. This technique is more fast, without solvent and effective when compared to hydrodistillation [14]. It was also evaluated on three aromatic herbs basil (*Ocimum basilicum*), garden mint (*Mentha crispa*), and thyme (*Thymus vulgaris*) where extraction time was significantly decreased from 4.5 h (hydro-distillation) to 30 min (SFME) [15]. This method was modified by many scientists. SFME method was modified by Wang et al. on dried *Cuminum cyminum* L. and *Zanthoxylum bungeanum* Maxim. by adding carbonyl iron powders (CIP) and mixed with the sample. CIP helps to reduce time (30 min) and microwave power (85 W) [16]. An attempt had been made to improve solvent-free extraction of essential oil using graphite powder, activated carbon powder. The effect was studied on *Illicium verum* Hook. f. and *Zingiber officinale* Rosc. [17]. Pressurized solvent-free microwave assisted extraction was used for extraction of phenolic compounds. The best extraction conditions were obtained, in a laboratory scale extractor of 50 mL filled with 4 g fresh berries, using a 1000 W microwave power applied during 50 s and repeated 5 cycles [18]. Solvent-free microwave extraction was modified by changing the flow of product toward the gravitation force. It is also known as Microwave dry-diffusion and gravity. It was better than hydrodistillation. The extraction performed in just 45 minutes with less energy and a clean process [19]. The extraction condition of SFME was optimized on *Elettaria cardamomum* (L.) using central composite design (CCD). The conditions such as time (min), power (W), humidity (%) was optimized by (CCD) and percentage yield (%) of was compared [20]. Optimization of SFME of pigeon pea leaves performed on an aliquot of 200 g plant materials that were wetted before extraction by soaking in water for 1 h [21]. Optimum parameters of SFME was performed on *S. chinensis* fruits and found ideal extraction time of 30 min, irradiation power 385 W and the moisture content of 68% respectively [22]. The quality of essential oil was also evaluated using Solvent-free microwave extraction method and compared with conventional method. It was found to be more effective than conventional method [23]. The effect of solvent-free microwave extraction was performed on several medicinal plants such as *Calamintha nepeta* [24], Basil leaves [25], *Dryopteris fragrans* [26], *Schisandra chinensis* [27]*, Cymbopogon winterianus* [28].


**Instrumentation Extraction conditions Results Reference**

Identification of optimum parameters was as follows; extraction time 30 min, irradiation power 385 W and the moisture content of *S. chinensis* fruits 68%,

The optimal parameters were extraction time 44 min, irradiation power 660 W, and humidity 68%, with extraction yield of 0.330 (%,

SFME exhibit shorter extraction times as compared to conventional method (30 min vs. 4.5 h) and better yields (0.13% vs. 0.11%)

This method exhibit shorter extraction time (15 min), cleaner feature (no solvent or water used) and extraction of valuable flavonoids (Isorhamnetin, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-rutinoside and quercetin 3-O-glucoside) at optimized

power (400 W)

It helps to extract more oxygenated compounds [22]

43

Solventless Extraction of Essential Oil http://dx.doi.org/10.5772/intechopen.72401

[21]

[25]

[32]

[33]

respectively

w/w)

A 100 g of *S. chinensis* fruits were moistened prior extraction by soaking in water then draining the excess of water. The extraction was continued until no distillate was obtained. The essential oil was collected, dried over anhydrous sodium sulfate and stored

200 g plant materials were wetted before extraction by soaking in a certain proportion of water for 1 h, and then removal the excess water. The wetted material was placed in the reaction flask and connected to a

150 g of fresh plant materials were placed in the reaction flask and heated by microwave irradiation with 400 W (50% power) for 30 min without adding any solvent or water. During the process, the vapor passed through the condenser outside the microwave cavity where it was condensed. Essential oil and water were simply separated by decantation. The essential oil was collected in amber vials, dried over anhydrous sodium sulfate and

400 g of sea buckthorn press cake was heated using a fix power density 1 W·g−1 without the addition of solvents or water. The crude extract was collected continuously in a graduated cylinder. The extraction was continued until no more extract was obtained or overheating was

30 g of dried *Lavandula angustifolia* was soaked in 20 mL distilled water at room temperature (25°C) for 1 h in order to hydrate the external layers of the plant material. The moistened plant material was placed in a flat-bottom flask combined with a Clevenger apparatus. The SFME process was performed for 35 min. The essential oils were collected in amber colored vials, dehydrated with anhydrous sodium sulfate, capped under nitrogen and kept at 4°C

at 0°C until analyzed

glass reaction flask

stored at 277 K

detected

Multimode microwave reactor, temperature Infrared sensor with maximum output power is 700 W, microwave rotating diffuser that ensures homogeneous microwave

The microwave-accelerated reaction system with multimode microwave reactor (2.45 GHz), IR temperature sensor, an electromagnetic stirrer, a time calculator controller, circulating

water-cooling system

Microwave oven (EMM-2007X, Electrolux, 20 l, the maximum delivered the power of 800 W) with a wave frequency of 2450 MHz. A round bottom flask with a capacity of 1000 ml was placed inside the oven and was connected to the three-way adapter and Liebig condenser through the hole. Then, the hole was closed with PTFE to prevent any loss of the heat inside

Milestone EOS-G microwave laboratory oven having a multimode microwave reactor (2.45 GHz) with a maximum delivered the power of 900 W. The extraction vessels are made from Pyrex and have a capacity of 1000 mL with a temperature sensor optic fiber which was inserted in the center of embedded plant material and also in the reactor above the

Microwave apparatus, 2450 MHz

with maximum power 1000 W and ACTE0 sensor for temperature monitoring. The power of the oven was 500 W for 10 min. The temperature was achieved at 95°C, and the extraction was carried out for

matrix

25 min

distribution

**Table 1.** List of medicinal plants and their identified essential oil evaluated under microwave assisted solvent-free extraction.



**Medicinal plants**

42 Green Chemistry

*Ocimum basilicum* L.

*Hippophae rhamnoides*

*Lavandula angustifolia*

*Calamintha nepeta*

extraction.

Reactor (500 mL), microwave oven, agitator, shielded noninvasive thermometry system, transformer with maximum output power is 800 W with 2450 MHz of microwave irradiation frequency (MIF).

The multi-mode reactor (2 × 800 W, 2450 MHz), rotating microwave diffuser, plasma coated PTFE cavity, circulating cooling system at 5°C

Microwave-accelerated reaction system (1000 W, 2450 MHz) multimode microwave reactor armed with a TFT multicolour liquid crystal screen, a power sensor (power range 0–1000 W), an infrared temperature sensor, a temperature controller and electromagnetic stirrer

**Identified essential oil Pharmacological** 

Sabinene, Octen 3 ol, β-Pinene, Heptanol, β-Myrcene, p-Cymene, Limonene, 1,8-Cineole, β-Ocimene, γ-Terpinene, Fenchone, Linalool, Camphor, Menthol, α-Terpineol, Methyl chavicol, Nerol, Neral, Geraniol, Geranial, α-Terpinenyl acetate, Neryl acetate, α-Copaene, Geranyl acetate, β-Bourbonene, β-Cubebene, β-Elemene, Methyl eugenol, β-Caryophyllene, α-Bergamotene, α-Humulene, β-Farnesene, Germacrene-D, γ-Cadinene, Δ-Cadinene, α-Bisabolene, β-Bisabolene, Spathulenol, Caryophyllene oxide, α-Cadinol

Isorhamnetin, isorhamnetin 3-O-glucoside, isorhamnetin 3-

1, 8-cineole, Camphor, Borneol, p-cymene, Limonene, Cryptone, isobornyl formate, cumin aldehyde, Valerianol, α-pinene

a-Thujene, a-Pinene, Sabinene, b-Pinene, D2-Carene, a-Terpinene, Camphene, (Z)-b-Ocimene, allo-Ocimene, Myrcene, Limonene, c-Terpinene, p-Cymene, Octan-3-ol, Eugenol, Geranyl acetone, Hexahydrofarnesylacetone, Octacosane, Phytol, Caryophyllene oxide, T-Cadinol, a-Cadinol, T-Muurolol, a-Copaene, b-Elemene, b-Cubebene, b-Bourbonene, a-Humulene, Caryophyllene, Germacrene-D, c-Cadinene, epi-Sesquiphellandrene, d-Cadinene, Menthyl acetate, Bornyl acetate, Menthol, Chrysanthenone,

Piperitenone, Piperitenone oxide, Isopulegonen, Pulegone, Piperitone, cis-Sabinene hydrate, 1,8-Cineole, Dihydrocarveol, trans-Sabinene hydrate, Menthone, Isomenthone, Terpinen-4-ol, a-Terpineol

**Table 1.** List of medicinal plants and their identified essential oil evaluated under microwave assisted solvent-free

**Instrumentation Extraction conditions Results Reference**

100 g of sample and 20 g of carbonyl iron powder (CIP) were added inside the reactor, stirred, heated (85 W) for 30 min at 100°C with speed of rotation (200 rpm), concentrated outside the microwave oven by a cooling system

250 g of Rosmarinus leaves were placed into the reactor without the addition of water or any solvent

200 g plant material was moistened prior to extraction by soaking in certain proportions of water (weight basis) for 1 h and then draining off the excess water. After that, the moistened materials were subjected to the microwave oven cavity and a condenser was used to collect the extracted essential oils in a pre-

setting procedure.

O-rutinoside and quercetin 3-O-glucoside

**activities**

N.A [25]

Anti-oxidant [32]

N.A [24]

[33]

[16]

[31]

[26]

Anti-bacterial activity

CIP helps to improve the microwave absorption capacity than water and is faster (30 min) than conventional method

Higher amounts of oxygenated monoterpenes were found as compared to conventional method

humidity 51% 16

A maximal extraction yield of 0.33% was achieved under optimal conditions of extraction time 34 min, irradiation power 520 W and **Reference**


**Table 2.** Instrumentation and extraction conditions of solvent-free extraction.

This modern method was transformed from laboratory scale to pilot and industrial scale [29]. List of Medicinal plants, their identified essential oil evaluated under Microwave assisted solvent-free extraction were represented in **Table 1**. Instrumentation and extraction conditions of solvent-free extraction were mentioned in **Table 2** (**Figures 1**–**3**).

**4. Conclusion**

**Figure 3.** Improved SFME [16].

**Figure 2.** Microwave hydro-diffusion and gravity (MHG) [30].

The term Solvent-less and solvent-free extraction have been used as synonymous with each other. Extraction of essential oil using these methods has a number of advantages such as fast action, cleanliness, green method, low energy output as compared to traditional extraction method. However, microwave extraction needs extra care before use as it may cause some negative effect on human health. There are many opportunities and modification possible in term of purification of essential oil by applying in combination with other extraction technique.

Solventless Extraction of Essential Oil http://dx.doi.org/10.5772/intechopen.72401 45

**Figure 1.** Solvent-free microwave extraction (SFME) [25, 30].

#### Solventless Extraction of Essential Oil http://dx.doi.org/10.5772/intechopen.72401 45

**Figure 2.** Microwave hydro-diffusion and gravity (MHG) [30].

#### **4. Conclusion**

This modern method was transformed from laboratory scale to pilot and industrial scale [29]. List of Medicinal plants, their identified essential oil evaluated under Microwave assisted solvent-free extraction were represented in **Table 1**. Instrumentation and extraction condi-

**Instrumentation Extraction conditions Results Reference**

Essential oil produced is lighter in color, higher yield, contains a cleaner, better purity and produced a stronger aroma compared to the essential oil produced from hydro-distillation

[12]

Fresh leaves of aromatic plants were grinded and to break them into smaller pieces and increasing the area of contact. Then, the grind leaves were put in a flask which was connected to another flask as a receiving flask. Firstly, the raw material was cooled down to a very low temperature to prevent decomposition and to avoid premature oil evaporation

tions of solvent-free extraction were mentioned in **Table 2** (**Figures 1**–**3**).

**Table 2.** Instrumentation and extraction conditions of solvent-free extraction.

**Figure 1.** Solvent-free microwave extraction (SFME) [25, 30].

Vacuum and nitrogen gas was applied on and off to remove air and replacing it with nitrogen in a closed system. At the end of the extraction process water and oil was separated and anhydrous sodium sulfate was used to dry

the excess water

44 Green Chemistry

The term Solvent-less and solvent-free extraction have been used as synonymous with each other. Extraction of essential oil using these methods has a number of advantages such as fast action, cleanliness, green method, low energy output as compared to traditional extraction method. However, microwave extraction needs extra care before use as it may cause some negative effect on human health. There are many opportunities and modification possible in term of purification of essential oil by applying in combination with other extraction technique.

## **Author details**

Muhammad Shahzad Aslam1,2\*, Muhammad Syarhabil Ahmad<sup>1</sup> and Sarwat Ali Raja<sup>3</sup> [11] Nasshorudin D, Ahmad MS, Mamat AS. Novel closed system extraction of essential oil:

Solventless Extraction of Essential Oil http://dx.doi.org/10.5772/intechopen.72401 47

[12] Ahmad MS, Nasshorudin D, Mamat AS. Novel closed system extraction of essential oil: Impact on yield and physical characterization muhammad. 2nd International

[13] Nasshorudin D, Ahmad MS, Mamat AS, Rosli S. Optimization study of *Chromalaena odorata* essential oil extracted using solventless extraction technique. AIP Conference

[14] Lucchesi ME, Chemat F, Smadja J. An original solvent free microwave extraction of essential oils from spices. Flavour and Fragrance Journal. Mar. 2004;**19**(2):134-138 [15] Lucchesi ME, Chemat F, Smadja J. Solvent-free microwave extraction of essential oil from aromatic herbs: Comparison with conventional hydro-distillation. Journal of

[16] Wang Z et al. Improved solvent-free microwave extraction of essential oil from dried *Cuminum cyminum* L. and *Zanthoxylum bungeanum* maxim. Journal of Chromatography.

[17] Wang Z et al. Rapid analysis of the essential oils from dried Illicium Verum Hook. f. And Zingiber Officinale Rosc. By improved solvent-free microwave extraction with three types of microwave-absorption medium. Analytical and Bioanalytical Chemistry. Nov.

[18] Michel T, Destandau E, Elfakir C.Evaluation of a simple and promising method for extraction of antioxidants from sea buckthorn (*Hippophaë rhamnoides* L.) berries: Pressurised

solvent-free microwave assisted extraction. *Food Chemistry*. 2011;**126**(3):1380-1386 [19] Farhat A, Fabiano-Tixier AS, Visinoni F, Romdhane M, Chemat F. A surprising method for green extraction of essential oil from dry spices: Microwave dry-diffusion and grav-

[20] Lucchesi ME, Smadja J, Bradshaw S, Louw W, Chemat F. Solvent free microwave extraction of Elletaria cardamomum L.: A multivariate study of a new technique for the extrac-

[21] Qi XL et al. Solvent-free microwave extraction of essential oil from pigeon pea leaves [*Cajanus cajan* (L.) Millsp.] and evaluation of its antimicrobial activity. Industrial Crops

[22] Ma CH, Yang L, Zu YG, Liu TT. Optimization of conditions of solvent-free microwave extraction and study on antioxidant capacity of essential oil from *Schisandra chinensis*

[23] Uysal B, Sozmen F, Aktas O, Oksal BS, Kose EO. Essential oil composition and antibacterial activity of the grapefruit (*Citrus Paradisi*. L) Peel essential oils obtained by solventfree microwave extraction: Comparison with hydrodistillation. International Journal of

tion of essential oil. Journal of Food Engineering. Apr. 2007;**79**(3):1079-1086

ity. Journal of Chromatography. A. 2010;**1217**(47):7345-7350

(Turcz.) Baill. Food Chemistry. 2012;**134**(4):2532-2539

Food Science and Technology. 2011;**46**(7):1455-1461

A green approach. American Journal of Biochemistry. 2016;**6**(6):145-148

Conference on Bioresource Technology. 2014;**75**:42-46

Chromatography. A. Jul. 2004;**1043**(2):323-327

Proceedings. 2015;**1660**

A. Jan. 2006;**1102**(1-2):11-17

and Products. 2014;**58**:322-328

2006;**386**(6):1863-1868

\*Address all correspondence to: aslammuhammadshahzad@gmail.com; shahzad.aslam@rlmc.edu.pk

1 School of Bioprocess Engineering, Universiti Malaysia Perlis (UniMAP), Arau, Perlis, Malaysia


#### **References**


[11] Nasshorudin D, Ahmad MS, Mamat AS. Novel closed system extraction of essential oil: A green approach. American Journal of Biochemistry. 2016;**6**(6):145-148

**Author details**

46 Green Chemistry

Malaysia

**References**

shahzad.aslam@rlmc.edu.pk

Press; pp. 552-557

therapy Research. 2002;**16**(4):301-308

Technologies. 2007;**8**(2):253-258

Control. 2014;**35**(1):109-116

2015;**5**(72):58449-58463

Muhammad Shahzad Aslam1,2\*, Muhammad Syarhabil Ahmad<sup>1</sup>

2 Rashid Latif College of Pharmacy, Lahore, Pakistan

\*Address all correspondence to: aslammuhammadshahzad@gmail.com;

3 Lahore Pharmacy College, (A project of LMDC), Lahore, Pakistan

food preservation. Journal of Food Science. 2014;**79**(7)

Chemico-Biological Interactions. 2009;**179**(2-3):160-168

1 School of Bioprocess Engineering, Universiti Malaysia Perlis (UniMAP), Arau, Perlis,

[1] Tongnuanchan P, Benjakul S. Essential oils: Extraction, bioactivities, and their uses for

[2] Cook CM, Lanaras T. The Encyclopedia of Food and Health. Chapter: Essential Oils: Isolation, Production and Uses. In: Cabballero B, Finglas P, Toldra F, editors. Academic

[3] Cavanagh HMA, Wilkinson JM. Biological activities of lavender essential oil. Phyto-

[4] Sharma PR et al. Anticancer activity of an essential oil from *Cymbopogon flexuosus*.

[5] Kumar Tyagi A et al. Eucalyptus essential oil as a natural food preservative: *In Vivo* and

[6] Gonçalves ND et al. Encapsulated thyme (*Thymus vulgaris*) essential oil used as a natural preservative in bakery product. Food Research International. 2017;**96**:154-160

[7] Tzortzakis NG, Economakis CD. Antifungal activity of lemongrass (*Cympopogon citratus* L.) essential oil against key postharvest pathogens. Innovative Food Science & Emerging

[8] Kedia A, Prakash B, Mishra PK, Dubey NK. Antifungal and antiaflatoxigenic properties of *Cuminum cyminum* (L.) seed essential oil and its efficacy as a preservative in stored

[9] Diao W-R, Hu Q-P, Zhang H, Xu J-G. Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (*Foeniculum vulgare* mill.). Food

[10] Majeed H et al. Essential oil encapsulations: Uses, procedures, and trends. RSC Advances.

commodities. International Journal of Food Microbiology. 2014;**168-169**:1-7

*In Vitro* antiyeast potential. BioMed Research International. 2014;**2014**:1-9

and Sarwat Ali Raja<sup>3</sup>


[24] Riela S et al. Effects of solvent-free microwave extraction on the chemical composition of essential oil of *Calamintha nepeta* (L.) Savi compared with the conventional production method. Journal of Separation Science. 2008;**31**(6-7):1110-1117

**Section 3**

**Pharmaceutical Green Chemistry**


**Pharmaceutical Green Chemistry**

[24] Riela S et al. Effects of solvent-free microwave extraction on the chemical composition of essential oil of *Calamintha nepeta* (L.) Savi compared with the conventional production

[25] Kusuma H, Putri D, Dewi I, Mahfud M. Solvent-free microwave extraction as the useful tool for extraction of edible essential oils. Chemistry & Chemical Technology.

[26] Li XJ et al. Solvent-free microwave extraction of essential oil from *Dryopteris fragrans* and

[27] Chen X, Zhang Y, Zu Y, Fu Y, Wang W. Composition and biological activities of the essential oil from *Schisandra chinensis* obtained by solvent-free microwave extraction.

[28] Paroul N et al. Solvent-free production of bioflavors by enzymatic esterification of citronella (*Cymbopogon winterianus*) essential oil. Applied Biochemistry and Biotechnology.

[29] Filly A, Fernandez X, Minuti M, Visinoni F, Cravotto G, Chemat F. Solvent-free microwave extraction of essential oil from aromatic herbs: From laboratory to pilot and indus-

[30] Li Y, Fabiano-Tixier AS, Vian MA, Chemat F. Solvent-free microwave extraction of bioactive compounds provides a tool for green analytical chemistry. TrAC, Trends in

[31] Okoh OO, Sadimenko AP, Afolayan AJ. Comparative evaluation of the antibacterial activities of the essential oils of *Rosmarinus officinalis* L. obtained by hydrodistillation and solvent free microwave extraction methods. Food Chemistry. 2010;**120**(1):308-312

[32] Périno-Issartier S, Zill-e-Huma, Abert-Vian M, Chemat F. Solvent free microwaveassisted extraction of antioxidants from sea buckthorn (*Hippophae rhamnoides*) food by-

[33] Azar A, Torabbeigi, Sharifan, Tehrani. Chemical composition and antibacterial activity of the essential oil of lavandula angustifolia isolated by solvent free microwave assisted extraction and hydrodistillation. Journal of Food Biosciences and Technology.

products. Food and Bioprocess Technology. 2011;**4**(6):1020-1028

evaluation of antioxidant activity. Food Chemistry. 2012;**133**(2):437-444

method. Journal of Separation Science. 2008;**31**(6-7):1110-1117

LWT- Food Science and Technology. 2011;**44**(10):2047-2052

trial scale. Food Chemistry. May 2014;**150**:193-198

Analytical Chemistry. 2013;**47**:1-11

2016;**10**(2):213-218

48 Green Chemistry

2012;**166**(1):13-21

2011;**1**:19-24

**Chapter 4**

**Provisional chapter**

**Green Chemistry and Synthesis of Anticancer**

**Green Chemistry and Synthesis of Anticancer** 

DOI: 10.5772/intechopen.70419

Green chemistry is a modern area of chemistry merged with chemical engineering methods. It highlighted the synthesis of molecules in a manner of using environment-friendly chemical reagents with low waste material for enhancing environmental performance which reduce the formation of hazard substances. Modern researches are trying to reduce the risk of human kind health and the environment of our world by doing magnificent work in the field of green chemistry. In the pharmaceutical field, green chemistry works very well with the formation of many drugs and it utilizes non-hazards, reproducible and environment-friendly solvents with low time and money costs by using catalyst, microwave, ultrasonic, solid phase and solvent-free synthesis. Until now, scientist has synthesized many anticancer molecules by using these modern green chemistry techniques. These compounds showed significant anticancer activities against many human cancer cell lines. In this chapter, we will cover different views and the recently published literature to summarize the role of green chemistry in the synthesis of anticancer

**Keywords:** green synthetic approaches, anticancer activity, synthesis of active molecules,

© 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,

© 2018 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.

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

Green chemistry is a modern way for the synthesis of organic compounds and designed different drugs under facile protocols, efficient conditions, environmentally benign and high yielding method of molecules with advantages over traditional organic synthetic methods. It usually reduces waste by-products, costs and develops environmentally friendly procedures.

Shagufta Perveen and Areej Mohammad Al-Taweel

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Shagufta Perveen and Areej Mohammad

http://dx.doi.org/10.5772/intechopen.70419

**Molecules**

**Abstract**

compounds.

cancer cell lines

**1. Introduction**

Al-Taweel

**Molecules**

**Provisional chapter**

#### **Green Chemistry and Synthesis of Anticancer Molecules Molecules**

**Green Chemistry and Synthesis of Anticancer** 

DOI: 10.5772/intechopen.70419

Shagufta Perveen and Areej Mohammad Al-Taweel Al-Taweel

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

Shagufta Perveen and Areej Mohammad

http://dx.doi.org/10.5772/intechopen.70419

#### **Abstract**

Green chemistry is a modern area of chemistry merged with chemical engineering methods. It highlighted the synthesis of molecules in a manner of using environment-friendly chemical reagents with low waste material for enhancing environmental performance which reduce the formation of hazard substances. Modern researches are trying to reduce the risk of human kind health and the environment of our world by doing magnificent work in the field of green chemistry. In the pharmaceutical field, green chemistry works very well with the formation of many drugs and it utilizes non-hazards, reproducible and environment-friendly solvents with low time and money costs by using catalyst, microwave, ultrasonic, solid phase and solvent-free synthesis. Until now, scientist has synthesized many anticancer molecules by using these modern green chemistry techniques. These compounds showed significant anticancer activities against many human cancer cell lines. In this chapter, we will cover different views and the recently published literature to summarize the role of green chemistry in the synthesis of anticancer compounds.

**Keywords:** green synthetic approaches, anticancer activity, synthesis of active molecules, cancer cell lines

#### **1. Introduction**

Green chemistry is a modern way for the synthesis of organic compounds and designed different drugs under facile protocols, efficient conditions, environmentally benign and high yielding method of molecules with advantages over traditional organic synthetic methods. It usually reduces waste by-products, costs and develops environmentally friendly procedures.

© 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 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.


Cellulose-supported

copper nanoparticlecatalyzed click

in the presence of K2CO3

to yield 4-*O*-propargylated benzaldehyde. In the next

step, 4-*O*-propargylated benzaldehyde was reacted with

substituted acetophenones via base-catalyzed Claisen-

Schmidt condensation to yield chalcones

Functionalized pyrazolopyridine derivatives via

Novel pyrazolo, pyridine derivatives (**6**)

Human A549 lung

[6]

adenocarcinoma, MCF-7

breast carcinoma cell line,

HCT-116 colon cancer, PC-3

prostate cancer

copper-promoted cyclization of pyridyl acetates and

benzonitriles in DMSO under argon atmosphere,

converted to corresponding pyrazolo[1, 5-a]pyridines

from commercially available aromatic nitriles and

various pyridyl acetates

Quinolone derivatives were synthesized by reacting

Pyrazolo[4,3-c] quinoline (5a-i,

Human MCF-7 breast and

[7]

A549 lung cancer

7a-b) and pyrano[3,2-c] quinoline

derivatives (**7**)

2,3-dihydro-8-nitro-4-quinolones with aromatic

aldehydes by pyrrolidine base-catalyzed condensation

reaction and were treated with hydrazine derivatives

under MW condition, which afforded pyrazolo quinoline

Synthetic route to barbituric acid derivatives substituted

Pyrimidine-2,4,6-trione derivatives (**8**)

HeLa cervical cancer and

[8]

3T3 mouse fibroblast cancer

at C5-position. Addition of barbituric acid analogous

into nitrostyrene, in water mediated by diethylamine as

base gave the target 5-monoalkylbarbiturates in excellent

derivatives in high yields

Facile protocol,

efficient and

environmentally

benign

yield

Simple, eco-friendly

To synthesize α,β-unsaturated carbonyl-based

α,β-unsaturated carbonyl-based

PC12 cancer

[9]

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 53

compounds (**9**)

compounds, Claisen-Schmidt condensation was used

between different ketones and suitable aryl aldehydes in

the presence of NaOH in ethanol

and efficient method

Microwave

conditions

in dry acetone under reflux

reaction in water

Copper-mediated

synthesis

**Reaction condition**

4-hydroxybenzaldehyde treated with propargyl bromide

**Class of compounds synthesized**

Chalcone-linked 1,2,3-triazoles (**5**)

**Cancer cell lines**

Human MCF-7, MIA-Pa-Ca-2,A549, HepG2 cancer

[5]


Eco-friendly one-pot

synthesis

**Reaction condition**

Mixture of methyl ketone, aldehyde, active methylene

cyanoacetamide, malononitrile, ethylcyanoacetate and

2 mL of glycol/ammonium acetate was added to the

reaction vessel and placed into MW reactor then allowed

to react under MW irradiation at 200–400

and 120°C for 6–8

by filtration and recrystallized from ethanol/DMF to

give pure amino-cyanopyridine and oxo-cyanopyridine

A mixture consisting of methyl salicylate and sodium

Salicyloyloxy and

Human breast

[2]

adenocarcinoma ER+,

MCF-7, estrogen

receptor negative breast

adenocarcinoma ER+,

MDA-MB-231, prostate

cancer PC-3 and normal

fetal lung fibroblasts MRC-5

cancer

Human A549 lung

[3]

adenocarcinoma

epithelial, MCF-7 breast

adenocarcinoma, HepG2

hepatocellular liver

carcinoma, DU145 prostate

cancer

Human HT-29 colon cancer

[4]

and MDA-MB breast cancer

and MRP1 inhibitory

activity using the insect cell

membrane

2-methoxybenzoyloxy androstane and

stigmastane derivatives (**2**)

was heated to 110°C.

for 30

min at 160–200°C using a 200

Chromatographic separation of crude mixture on silica

gel column gave the pure products

One-pot synthesis

Reaction of 2-chloro-3-chloromethyl-quinoline

Quinoline, triazole and

dihydroquinoline (**3**)

with terminal alkyne in the presence of KI, NaN3

and precatalyst copper(II)sulfate in combination

with Na-ascorbate was examined in water at room

temperature

Microwave-assisted

Ethyl/methyl acetoacetate and an aldehyde were taken

4-alkyl/aryl-3,5-bis(carboethoxy/

carbomethoxy)-1,4-dihydro-2,6-

imethylpyridines (**4**)

into a beaker and dissolved in minimum quantity of

dimethylformamide. To this solution, ammonium

acetate was added. Reaction mixture was subjected to

rate of 60 s each in a microwave oven. After completion

of the reaction on TLC, the resultant product was filtered,

washed with chill water and recrystallized

W for 2–6 min, with a pulse

microwave irradiation at 480

synthesis

W MW source.

was completed after 5–10 min, mixture was irradiated

When the reaction with sodium

derivatives

Microwave-assisted

synthesis

min. The compound was collected

W power

**Class of compounds synthesized**

Novel cyanopyridine derivatives (**1**)

**Cancer cell lines**

Human liver HepG2, colon

[1]

52 Green Chemistry

HCT-116, breast MCF-7

cancer


Catalyst-free, green

approach

**Reaction condition**

Mixture of 2-(1H-pyrrol-1-yl) aniline and isatin in EtOH

was refluxed at 80°C for 6 h. The progress of reaction was

On the completion, it cooled to room

monitored by TLC.

temperature and then precipitated product was filtered,

washed with EtOH and dried by rotavapor to afford

Benzaldehyde was reacted with morpholine and

Morpholine-pyrazolidine

derivatives (**17**)

HepG2 liver, HeLa cervical and MCF-7 breast cancer

[17]

2,4-dinitrophenyl hydrazine in the presence of a

chiral pyrrolidine-based catalyst in EtOH.

step, compounds were reacted with cinnamaldehyde

in the presence of chiral catalyst in toluene at room

temperature for 5 h. Compounds were obtained in

excellent yields (88–96%)

A mixture of three components, thiosemicarbazide,

New bithiazolyl hydrazones (**18**)

cancers

MCF-7, HCT116 and THP-1

[18]

5-acyl thiazoles and phenacyl chlorides, was dissolved

in freshly prepared non-volatile organic solvent, DIPEAc

and the solution was stirred at room temperature for

min. Then, the products were isolated with excellent

30 yields, 82–96%

One-pot synthesis

Series of *N*-(aminosulfonyl)-4-podophyllotoxin

N-(aminosulfonyl)-4-podophyllotoxin

Human tumor HeLa, A-549,

[19]

HCT-8 and HepG2, human

fetal lung fibroblast WI-38

cancer cells

carbamates (**19**)

carbamates were synthesized with amines and

*N*-(chlorosulfonyl)-4-podophyllotoxin carbamate dry

CH2Cl2 via Burgess-type intermediate, which generated

in situ by reaction of PPT and chlorosulfonyl isocyanate

CSI in the presence of pyridine

Tryptamine, 2-hydroxy-4,6-dimethylpyrimidine and

Bacillamide analogues (**20**)

Human colorectal

[20]

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419

tumor HCT-116, breast

adenocarcinoma

MDA-MB-231 and immune

system JURKAT cancer

appropriate thiazole-4-carboxylate were homogenized

and then heated at 100–105°C for 5–6 h. The reaction

mixture was concentrated and the crude product so

obtained was crystallized from EtOH

Amine and ferulic acid were mixed together in 1:1 ratio

Ferulic acid amide derivatives (**21**)

Human breast

[21]

MDA-MB-231 and MCF-7,

cervical HeLa, lung A549

and liver HepG2

55

for mono-amide and 2:1 molar ratio for bisamide. The

reaction mixture was irradiated in microwave at 180–450

min. The reaction progress was monitored

Products were obtained and purified by

Watt for 3–7

by TLC. crystallization

Microwave-assisted

synthesis

One-pot solvent-free

synthesis

Novel one-pot

cyclocondensation

In the next

pure compound

Simple and

convenient one-pot

four-component

synthesis

**Class of compounds synthesized**

Pyrrolospirooxindole derivatives (1**6**)

DU-145

Human prostate cancer

[16]

**Cancer cell lines**


Synthesis by using

green solvents

**Reaction condition**

2,3-Dihydrophthalazine-1,4-dione, 5,5-dimethyl

clohexane-1,3-dione, aldehyde and p-sulfonic acid

calix[4]arene were dissolved in EtOAc. The mixture

was irradiated in a MW reactor for 10

reaction was cooled to room temperature and then water

was added. The mixture was placed in a freezer at 20°C

to form the product

One-pot reaction

Mixture of 2-thioxoimidazolidin-4-one and sodium

ethoxide in EtOH was refluxed for 30

CS2 was added and the reaction mixture was stirred at

room temperature for 1 h. After evaporation of solution,

the solid product was recrystallized from EtOH to give

compound in 70–75% yield

One-pot synthesis of 5-amino-2-(4-chlorophenyl)-

7-substituted phenyl-8,8a-dihydro-7H-3,4)

thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives

from three component reactions of 5-(4-chlorophenyl)-

1,3,4-thiadiazol-2 amine, aromatic aldehydes and

malononitrile in the presence of NaOH under reflux and

ultrasonic irradiation

Microwave irradiation of mixture of aldehyde and

1,2-phenylenediamine at 80°C, 150

 W for 5 Na2S2O5 for oxidation, the product 1H-benzo[d]

imidazol-2-yl)-6,7,8-trimethoxynaphthalen-1-ol was

isolated in excellent 95% yield

These reactions were carried out using catechol,

Novel pyrimidobenzothiazoles

Human HepG2 cancer

[14]

and catechol thioethers (**14**)

2,3-dihydro-2-thioxopyrimidin-4(1H)-ones and enzyme

laccase, phosphate buffer pH 6 and EtOH with nice

A solution of α-halocarbonyl derivative in dry acetone

Aryl-hydrazinyl-1,3-selenazole

Human leukemia cell lines

[15]

CCRF-CEM and HL60

and carcinoma cell lines

MDA-MB231, HCT116 and

U87MG

andaroyl-hydrazonyl-1,3-selenazoles

was added to the solution of benzylidene hydrazine

carboseleno amide derivative in DMF.

mixture was stirred at room temperature for 1 day and

then neutralized with NaHCO3. The precipitate was

filtered and then recrystallized from EtOH

The reaction

(**15**)

yields 95%

Microwave-assisted

Hantzsch type

condensation

reactions

Laccase-catalyzed

green synthesis

min using

2-quinolizinylbenzimidazole

Human breast MCF-7

[13]

cancer

and 2-naphthalylbenzimidazole

derivatives (**13**)

Microwave-assisted

synthesis

5-amino-2-(4-chlorophenyl)-

MCF-7, K562, HeLa and

[12]

PC-3 cancer

7-substituted phenyl-8,8adihydro-7H-(1,3,4)thiadiazolo (3,2-α)pyrimidine-6-carbonitrile

derivatives (**12**)

One-pot ultrasoundpromoted synthesis

min. After cooling,

Novel 2-thioxoimidazolidin-4-one

and benzothiazole thiolate salts (**11**)

min at 130°C.

 The

**Class of compounds synthesized**

Phthalazine-triones: Calix[4]arene (**10**)

**Cancer cell lines**

Human tumor U251 glioma,

[10]

54 Green Chemistry

MCF7 breast NCIADR/

RES multiple drug-resistant

ovarian, 786-0 renal, NCI-H460 lung, non-small cells,

PC-3 prostate, OVCAR-03

ovarian, HT-29 colon and

K562 leukemia cancer

MCF-7 breast carcinoma

[11]

cancer


**Table 1.** Anticancer molecules by green synthesis. This chemistry surrounds a series of modern techniques for synthesizing bioactive com

Cancer is a disease generated by uncontrolled cell growth in the body. There are many pro

gresses for cancer treatment but it remains mostly common cause of human death. The num

ber of cancer patients is increasing significantly worldwide, especially in developed countries. According to the global oncology trend report (2015), global spending on cancer medications rose 10.3% in 2014 to \$100 billion from \$ 75 billion in 2009. Therefore, there is a quick and urgent need of systematic approach to the development of new chemotherapeutic agents with superior efficacy, lower toxicity as well as better selectivity. The methods used in green chem

istry organic synthesis of molecules are playing wide role for designing the anticancer drugs. In this chapter, we discuss the most recent literature on green synthesis of different molecules

Green chemistry is one of the valuable concepts for the development of new, more effective, solvent-free less toxic, environmentally friendly and cost-efficient methods for the synthe

sis of different anticancer molecules. There are many developments for the environmentally friendly approaches for the synthesis of biologically active molecules such as microwaveassisted synthesis, one-pot synthesis, solvent-free synthesis, enzyme-catalyzed synthesis, solid phase synthesis, ultrasound promoted and catalyst-free synthesis. Herein, we are dis

cussing some recently published cytotoxic molecules, which have been synthesized by differ

**1**).

reduce environmental hazards and to minimize ecological risks.

and their anticancer potential on different human cancer cell lines (**Table**

**2. Green synthesis of different anticancer molecules**

ent green synthesis approaches (**Figure**

**Figure 1.** 3-cyano pyridine derivatives.

pounds, such as microwave-assisted synthesis, solid phase supported solvent-free synthesis, reaction with organocatalyst, one-pot multicomponent reactions and sonochemical synthesis, using ionic liquids techniques. Pharmaceutical companies are also improving chemicals to


57







**1**).

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 This chemistry surrounds a series of modern techniques for synthesizing bioactive compounds, such as microwave-assisted synthesis, solid phase supported solvent-free synthesis, reaction with organocatalyst, one-pot multicomponent reactions and sonochemical synthesis, using ionic liquids techniques. Pharmaceutical companies are also improving chemicals to reduce environmental hazards and to minimize ecological risks.

Cancer is a disease generated by uncontrolled cell growth in the body. There are many progresses for cancer treatment but it remains mostly common cause of human death. The number of cancer patients is increasing significantly worldwide, especially in developed countries. According to the global oncology trend report (2015), global spending on cancer medications rose 10.3% in 2014 to \$100 billion from \$ 75 billion in 2009. Therefore, there is a quick and urgent need of systematic approach to the development of new chemotherapeutic agents with superior efficacy, lower toxicity as well as better selectivity. The methods used in green chemistry organic synthesis of molecules are playing wide role for designing the anticancer drugs. In this chapter, we discuss the most recent literature on green synthesis of different molecules and their anticancer potential on different human cancer cell lines (**Table 1**).

#### **2. Green synthesis of different anticancer molecules**

Green chemistry is one of the valuable concepts for the development of new, more effective, solvent-free less toxic, environmentally friendly and cost-efficient methods for the synthesis of different anticancer molecules. There are many developments for the environmentally friendly approaches for the synthesis of biologically active molecules such as microwaveassisted synthesis, one-pot synthesis, solvent-free synthesis, enzyme-catalyzed synthesis, solid phase synthesis, ultrasound promoted and catalyst-free synthesis. Herein, we are discussing some recently published cytotoxic molecules, which have been synthesized by different green synthesis approaches (**Figure 1**).

**Figure 1.** 3-cyano pyridine derivatives.

**Method use** Catalyst under

solvent-free

conditions

15

**Reaction condition**

The aromatic aldehyde, 2-hydroxy-1,4-naphthoquinone

and 2-naphthol grinded in a mortar for 5

InCl3 was added and the reaction mixture was grinded

min again then placed in a sealed tube and kept in an

oven at 120°C for 3 h. The resulting crude was purified

by chromatography

**Table 1.**

Anticancer molecules by green synthesis.

min. Then,

**Class of compounds synthesized**

Dibenzo anthracenes (**22**)

**Cancer cell lines**

HEL human

erythroleukemia and

MCF7 breast cancer

**Refs.**

[22]

56 Green Chemistry

#### **2.1. One-pot synthesis of 3-cyano pyridine derivatives**

Novel series of 3-cyano pyridine type derivatives were synthesized and their cytotoxic activity was evaluated against many human MCF-7, HCT-116 and HepG-2 cancer cell lines. Most of the compounds showed good-to-moderate activity against HepG2 and HCT-116 cell lines, whereas only few compounds showed significant cytotoxic activity against MCF-7 breast cancer cell line (**Figure 1**) [1].

#### **2.2. Microwave-assisted solvent-free synthesis of stigmastane derivatives**

The microwave-assisted synthesis in most cases was more successful regarding to the reaction time and the yields of product. These reactions are more environmentally friendly too, compared to the conventional synthetic methods. In this research, a convenient simple microwave-assisted solvent-free synthesis of 2-methoxybenzoyloxy androstane, salicyloyloxy stigmastane derivatives from methyl salicylate and appropriate steroidal precursors has done. 2-Methoxybenzoyl ester exhibited significant cytotoxic activity against MDA-MB-231 cells. Most of the compounds strongly inhibited growth of PC-3 cells, whereas salicyloyloxy stigmastane derivative showed the best inhibition potency (**Figure 2**) [2].

**2.5. Cellulose-supported copper nanoparticle-catalyzed synthesis of chalcone derivatives**

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 59

Chalcone-linked 1,2,3-triazole derivatives were synthesized in water by cellulose-supported copper nanoparticle-catalyzed click reaction. All the products were subjected to MTT cytotoxicity assay against four human cancer cell lines A549, MCF-7, HepG2 and MIA-Pa-Ca-2 for testing their anticancer potential. Few compounds were found to be most active against all cancer cell lines and showed better activity when compared to reference drug (**Figure 5**) [5].

Some novel pyrazolo pyridine type compounds were synthesized by facile procedures and showed significant cytotoxic potential on different human cancer cell lines. They revealed various cancer cell lines (HCT-116, A549, MCF-7, PC-3) determined by SRB assay (**Figure 6**) [6].

A new class of pyrazolo[4,3-c]quinoline and pyrano[3,2-c]quinoline analogues was synthesized in good yields by microwave conditions. For enhancing the yield of products, multicomponent one-pot synthesis has been developed. The cytotoxicity of these compounds was also evaluated against MCF-7 and A549 cancer cell lines. Most of the compounds displayed

**2.6. Copper-mediated synthesis of pyrazolo pyridine derivatives**

**Figure 3.** Synthesis of polyazaheterocycles.

**2.7. Microwave-assisted synthesis of quinoline analogues**

**Figure 4.** Synthesis of carboethoxy/carbomethoxy derivatives.

moderate-to-good anticancer activity against these cell lines (**Figure 7**) [7].

#### **2.3. One-pot synthesis of polyazaheterocycles in water**

Synthesis of these polyazaheterocycles was carried out by green synthetic strategy that involved one-pot azidation and CuAAC under mild conditions in water. Many compounds were synthesized and evaluated for their cytotoxic effects against four human cancer cell lines, including A549 (lung), MCF-7 (breast), HepG2 (hepatocellular) and DU145 (prostate). Some of the compounds showed strong activities against A549 cancer cells (**Figure 3**) [3].

#### **2.4. Microwave irradiated one-pot synthesis of carboethoxy/carbomethoxy derivatives**

Fourteen carboethoxy/carbomethoxy derivatives have been synthesized by conventional and microwave irradiation method from a one-pot three-component reaction mixture, consisting of, alkyl acetoacetate, aldehyde and ammonium acetate. The synthesized products have been evaluated for their cytotoxic activity against MDA-MB (breast) and HT-29 (colon) human cancer cell lines. Few compounds exhibit some degree of cytotoxicity and it was low when compared with standard (**Figure 4**) [4].

**Figure 2.** Synthesis of stigmastane derivatives.

**Figure 3.** Synthesis of polyazaheterocycles.

**2.1. One-pot synthesis of 3-cyano pyridine derivatives**

the best inhibition potency (**Figure 2**) [2].

compared with standard (**Figure 4**) [4].

**Figure 2.** Synthesis of stigmastane derivatives.

**2.3. One-pot synthesis of polyazaheterocycles in water**

(**Figure 1**) [1].

58 Green Chemistry

Novel series of 3-cyano pyridine type derivatives were synthesized and their cytotoxic activity was evaluated against many human MCF-7, HCT-116 and HepG-2 cancer cell lines. Most of the compounds showed good-to-moderate activity against HepG2 and HCT-116 cell lines, whereas only few compounds showed significant cytotoxic activity against MCF-7 breast cancer cell line

The microwave-assisted synthesis in most cases was more successful regarding to the reaction time and the yields of product. These reactions are more environmentally friendly too, compared to the conventional synthetic methods. In this research, a convenient simple microwave-assisted solvent-free synthesis of 2-methoxybenzoyloxy androstane, salicyloyloxy stigmastane derivatives from methyl salicylate and appropriate steroidal precursors has done. 2-Methoxybenzoyl ester exhibited significant cytotoxic activity against MDA-MB-231 cells. Most of the compounds strongly inhibited growth of PC-3 cells, whereas salicyloyloxy stigmastane derivative showed

Synthesis of these polyazaheterocycles was carried out by green synthetic strategy that involved one-pot azidation and CuAAC under mild conditions in water. Many compounds were synthesized and evaluated for their cytotoxic effects against four human cancer cell lines, including A549 (lung), MCF-7 (breast), HepG2 (hepatocellular) and DU145 (prostate). Some of the compounds showed strong activities against A549 cancer cells (**Figure 3**) [3].

**2.4. Microwave irradiated one-pot synthesis of carboethoxy/carbomethoxy derivatives**

Fourteen carboethoxy/carbomethoxy derivatives have been synthesized by conventional and microwave irradiation method from a one-pot three-component reaction mixture, consisting of, alkyl acetoacetate, aldehyde and ammonium acetate. The synthesized products have been evaluated for their cytotoxic activity against MDA-MB (breast) and HT-29 (colon) human cancer cell lines. Few compounds exhibit some degree of cytotoxicity and it was low when

**2.2. Microwave-assisted solvent-free synthesis of stigmastane derivatives**

#### **2.5. Cellulose-supported copper nanoparticle-catalyzed synthesis of chalcone derivatives**

Chalcone-linked 1,2,3-triazole derivatives were synthesized in water by cellulose-supported copper nanoparticle-catalyzed click reaction. All the products were subjected to MTT cytotoxicity assay against four human cancer cell lines A549, MCF-7, HepG2 and MIA-Pa-Ca-2 for testing their anticancer potential. Few compounds were found to be most active against all cancer cell lines and showed better activity when compared to reference drug (**Figure 5**) [5].

#### **2.6. Copper-mediated synthesis of pyrazolo pyridine derivatives**

Some novel pyrazolo pyridine type compounds were synthesized by facile procedures and showed significant cytotoxic potential on different human cancer cell lines. They revealed various cancer cell lines (HCT-116, A549, MCF-7, PC-3) determined by SRB assay (**Figure 6**) [6].

#### **2.7. Microwave-assisted synthesis of quinoline analogues**

A new class of pyrazolo[4,3-c]quinoline and pyrano[3,2-c]quinoline analogues was synthesized in good yields by microwave conditions. For enhancing the yield of products, multicomponent one-pot synthesis has been developed. The cytotoxicity of these compounds was also evaluated against MCF-7 and A549 cancer cell lines. Most of the compounds displayed moderate-to-good anticancer activity against these cell lines (**Figure 7**) [7].

**Figure 4.** Synthesis of carboethoxy/carbomethoxy derivatives.

**2.8. Facile protocol, efficient and environmentally benign synthesis of cycloheximide**

activity against the standard cycloheximide (**Figure 8**) [8].

**Figure 8.** Synthesis of quinoline cycloheximide.

In this research, they describe a facile and efficient protocol and environmentally benign for the synthesis of C5-substituted barbiturate acid in water. The synthesized compounds tested for different assay and provided promising results against a-glucosidase inhibitor. The cytotoxic activity of compound against 3T3 cell resulted that compounds showed significant to weak

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 61

**2.9. Simple, eco-friendly and efficient synthesis of α,β-unsaturated carbonyl compounds**

showed strong protective effect against PC12 cell line (**Figure 9**) [9].

**Figure 9.** Synthesis of α,β-unsaturated carbonyl-based compounds.

A novel series of carbonyl compounds was synthesized by environment-friendly, simple and efficient method. Compounds were tested for cytotoxicity. All strong antioxidant compounds

**Figure 5.** Synthesis of chalcone derivatives.

**Figure 6.** Synthesis of pyridine derivatives.

**Figure 7.** Synthesis of quinoline analogues.

#### **2.8. Facile protocol, efficient and environmentally benign synthesis of cycloheximide**

In this research, they describe a facile and efficient protocol and environmentally benign for the synthesis of C5-substituted barbiturate acid in water. The synthesized compounds tested for different assay and provided promising results against a-glucosidase inhibitor. The cytotoxic activity of compound against 3T3 cell resulted that compounds showed significant to weak activity against the standard cycloheximide (**Figure 8**) [8].

#### **2.9. Simple, eco-friendly and efficient synthesis of α,β-unsaturated carbonyl compounds**

A novel series of carbonyl compounds was synthesized by environment-friendly, simple and efficient method. Compounds were tested for cytotoxicity. All strong antioxidant compounds showed strong protective effect against PC12 cell line (**Figure 9**) [9].

**Figure 9.** Synthesis of α,β-unsaturated carbonyl-based compounds.

**Figure 7.** Synthesis of quinoline analogues.

**Figure 5.** Synthesis of chalcone derivatives.

60 Green Chemistry

**Figure 6.** Synthesis of pyridine derivatives.

#### **2.10. Green methodology synthesis of 2H-indazolo[2,1-b]phthalazine-trione derivatives**

**2.12. Green synthesis of pyrimidine-6-carbonitrile derivatives**

**2.13. Microwave-assisted synthesis of benzimidazole derivatives**

**Figure 13.** Synthesis of pyrimidine-6-carbonitrile derivatives.

pounds were found to be as active as standard Tamoxifen (**Figure 13**) [13].

and MCF-7 cancer cell lines (**Figure 12**) [12].

**Figure 12.** Synthesis of pyrimidine-6-carbonitrile derivatives.

This is a green synthetic approach for the formation of antitumor active 5-amino-2-(4 chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α) pyrimidine-6-carbonitrile. This protocol is extendable to a wide variety of many substrates. The advantages are the use of eco-friendly catalyst, reduced time, simple work-up process, ease of isolation and high yield of product. One compound was found to have the highest GI50 value for PC-3, HeLa, K562

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 63

Twelve 2-quinolizinylbenzimidazole and 2-naphthalylbenzimidazole type compounds have been synthesized under MW microwave condition. These compounds were tested for cytotoxicity against human breast cancer cell line MCF-7. The results showed that some com-

An efficient green method was used for the synthesis of 2H-indazolo[2,1-b]phthalazine-trione derivatives. Many compounds were obtained in good yields within 10 min. Among all tested cell lines, K562 leukemia cell line was most sensitive (**Figure 10**) [10].

**Figure 10.** Synthesis of phthalazine-trione derivatives.

#### **2.11. Green synthesis of thioxoimidazolidin and benzothiazole derivatives**

A series of 2-thioxoimidazolidin-4-one and benzothiazole thioglycosides were synthesized by one-pot reaction. The cytotoxic activity of compound was evaluated against MCF-7 breast cell and it showed high-to-moderate anticancer activities (**Figure 11**) [11].

**Figure 11.** Synthesis of thioxoimidazolidin and benzothiazole derivatives.

#### **2.12. Green synthesis of pyrimidine-6-carbonitrile derivatives**

**2.10. Green methodology synthesis of 2H-indazolo[2,1-b]phthalazine-trione derivatives**

cell lines, K562 leukemia cell line was most sensitive (**Figure 10**) [10].

**2.11. Green synthesis of thioxoimidazolidin and benzothiazole derivatives**

and it showed high-to-moderate anticancer activities (**Figure 11**) [11].

**Figure 10.** Synthesis of phthalazine-trione derivatives.

62 Green Chemistry

**Figure 11.** Synthesis of thioxoimidazolidin and benzothiazole derivatives.

A series of 2-thioxoimidazolidin-4-one and benzothiazole thioglycosides were synthesized by one-pot reaction. The cytotoxic activity of compound was evaluated against MCF-7 breast cell

An efficient green method was used for the synthesis of 2H-indazolo[2,1-b]phthalazine-trione derivatives. Many compounds were obtained in good yields within 10 min. Among all tested This is a green synthetic approach for the formation of antitumor active 5-amino-2-(4 chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α) pyrimidine-6-carbonitrile. This protocol is extendable to a wide variety of many substrates. The advantages are the use of eco-friendly catalyst, reduced time, simple work-up process, ease of isolation and high yield of product. One compound was found to have the highest GI50 value for PC-3, HeLa, K562 and MCF-7 cancer cell lines (**Figure 12**) [12].

**Figure 12.** Synthesis of pyrimidine-6-carbonitrile derivatives.

#### **2.13. Microwave-assisted synthesis of benzimidazole derivatives**

Twelve 2-quinolizinylbenzimidazole and 2-naphthalylbenzimidazole type compounds have been synthesized under MW microwave condition. These compounds were tested for cytotoxicity against human breast cancer cell line MCF-7. The results showed that some compounds were found to be as active as standard Tamoxifen (**Figure 13**) [13].

**Figure 13.** Synthesis of pyrimidine-6-carbonitrile derivatives.

#### **2.14. Enzyme laccase-catalyzed green synthesis of pyrimidobenzothiazoles**

This is a newly developed method for the synthesis of pyrimidobenzothiazoles and catechol thioethers, and it addressed many of the principles of green chemistry. These reactions were catalyzed by laccase enzyme and transformations were completely safe and non-toxic aerial oxygen as the sole oxidant. This reaction delivers the products in an excellent yield. Among all tested compounds, few compounds showed moderate-to-good activity against HepG2 cell line (**Figure 14**) [14].

**2.17. One-pot four-component synthesis of morpholine-connected pyrazolidine** 

showed significant cytotoxicity against tested cells (**Figure 17**) [17].

**2.18. Novel one-pot cyclocondensation**

**Figure 16.** Synthesis of spiro[indoline-3,4′-pyrrolo(1,2-a)quinoxalin.

**Figure 15.** Synthesis of 1,3-selenazole derivatives.

A simple and convenient one-pot four component reaction of morpholine connected with pyrazolidine derivatives was developed using metal-free catalysis. Cytotoxicity was evaluated using HeLa (cervical), HepG2 (liver) and MCF-7 (breast) cancer cell lines, and compounds

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 65

Bithiazolyl hydrazones have been synthesized by one-pot cyclocondensation reaction in freshly prepared ionic liquid at room temperature. Compounds have been evaluated for anti-

tubercular activity and showed potent antitubercular activity (**Figure 18**) [18].

**derivatives**

**Figure 14.** Synthesis of pyrimidobenzothiazoles.

#### **2.15. Microwave-assisted synthesis of 1,3-selenazole derivatives**

Synthesis of new 1,3-selenazole derivatives has been done by MW-assisted Hantzsch condensation reactions. Compound were screened for anti-proliferative effects against leukemia cell lines (HL60 and CCRF-CEM) and carcinoma cell lines (HCT116, MDA-MB231 and U87MG) and it gave moderate cytotoxicity against all tested cell lines (**Figure 15**) [15].

#### **2.16. Environment-friendly synthesis of 5′H-spiro[indoline-3,4′-pyrrolo(1,2-a) quinoxalin]-2-ones**

A very simple-to-perform, efficient, mild and environment-friendly benign formation of 5′H-spiro[indoline-3,4′-pyrrolo(1,2-a)quinoxalin]-2-ones has been developed without any catalysts. This method includes simplicity of operation, clean reaction, no side products and good yields. Purification of product is very simple, involving a filtration and washing. The synthesized compound with piperonyl substitution on 5-chloroisatin nitrogen showed highest cytotoxicity. Its IC50 values are comparable to that of the standard doxorubicin (**Figure 16**) [16].

**Figure 15.** Synthesis of 1,3-selenazole derivatives.

**2.14. Enzyme laccase-catalyzed green synthesis of pyrimidobenzothiazoles**

**2.15. Microwave-assisted synthesis of 1,3-selenazole derivatives**

and it gave moderate cytotoxicity against all tested cell lines (**Figure 15**) [15].

**2.16. Environment-friendly synthesis of 5′H-spiro[indoline-3,4′-pyrrolo(1,2-a)**

Synthesis of new 1,3-selenazole derivatives has been done by MW-assisted Hantzsch condensation reactions. Compound were screened for anti-proliferative effects against leukemia cell lines (HL60 and CCRF-CEM) and carcinoma cell lines (HCT116, MDA-MB231 and U87MG)

A very simple-to-perform, efficient, mild and environment-friendly benign formation of 5′H-spiro[indoline-3,4′-pyrrolo(1,2-a)quinoxalin]-2-ones has been developed without any catalysts. This method includes simplicity of operation, clean reaction, no side products and good yields. Purification of product is very simple, involving a filtration and washing. The synthesized compound with piperonyl substitution on 5-chloroisatin nitrogen showed highest cytotoxicity. Its IC50 values are comparable to that of the standard doxorubicin

line (**Figure 14**) [14].

64 Green Chemistry

**quinoxalin]-2-ones**

**Figure 14.** Synthesis of pyrimidobenzothiazoles.

(**Figure 16**) [16].

This is a newly developed method for the synthesis of pyrimidobenzothiazoles and catechol thioethers, and it addressed many of the principles of green chemistry. These reactions were catalyzed by laccase enzyme and transformations were completely safe and non-toxic aerial oxygen as the sole oxidant. This reaction delivers the products in an excellent yield. Among all tested compounds, few compounds showed moderate-to-good activity against HepG2 cell

#### **2.17. One-pot four-component synthesis of morpholine-connected pyrazolidine derivatives**

A simple and convenient one-pot four component reaction of morpholine connected with pyrazolidine derivatives was developed using metal-free catalysis. Cytotoxicity was evaluated using HeLa (cervical), HepG2 (liver) and MCF-7 (breast) cancer cell lines, and compounds showed significant cytotoxicity against tested cells (**Figure 17**) [17].

#### **2.18. Novel one-pot cyclocondensation**

Bithiazolyl hydrazones have been synthesized by one-pot cyclocondensation reaction in freshly prepared ionic liquid at room temperature. Compounds have been evaluated for antitubercular activity and showed potent antitubercular activity (**Figure 18**) [18].

**Figure 16.** Synthesis of spiro[indoline-3,4′-pyrrolo(1,2-a)quinoxalin.

**2.19. One-pot synthesis of carbamates**

**2.20. Eco-friendly synthesis of bacillamide**

**Figure 19.** Synthesis of N-(aminosulfonyl)-4-podophyllotoxin carbamates.

doxorubicin (**Figure 20**) [20].

One-pot synthesis of N-(aminosulfonyl)-4-podophyllotoxin carbamates has been done and it showed promising cytotoxic activities. Most effective compound induced HeLa cells cycle arrest in G2/M phase, leading to apoptosis, and activation of cdc2, cyclinB1, p53 and ROS and inhibits polymerization of tubulin and microtubule. These results suggest that these synthesized compounds have strong potential for development as cytotoxic agents (**Figure 19**) [19].

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 67

It is an efficient, advanced and eco-friendly route for synthesis of bacillamide analogues through a two-step solvent-free synthesis. Compounds exhibit potent cytotoxic activity against three HCT-116, MDA-MD-231 and JURKATs cancer cell lines and compared with

**Figure 17.** Synthesis of morpholine-connected pyrazolidine.

**Figure 18.** Synthesis of bithiazolyl hydrazones.

#### **2.19. One-pot synthesis of carbamates**

One-pot synthesis of N-(aminosulfonyl)-4-podophyllotoxin carbamates has been done and it showed promising cytotoxic activities. Most effective compound induced HeLa cells cycle arrest in G2/M phase, leading to apoptosis, and activation of cdc2, cyclinB1, p53 and ROS and inhibits polymerization of tubulin and microtubule. These results suggest that these synthesized compounds have strong potential for development as cytotoxic agents (**Figure 19**) [19].

#### **2.20. Eco-friendly synthesis of bacillamide**

It is an efficient, advanced and eco-friendly route for synthesis of bacillamide analogues through a two-step solvent-free synthesis. Compounds exhibit potent cytotoxic activity against three HCT-116, MDA-MD-231 and JURKATs cancer cell lines and compared with doxorubicin (**Figure 20**) [20].

**Figure 19.** Synthesis of N-(aminosulfonyl)-4-podophyllotoxin carbamates.

**Figure 18.** Synthesis of bithiazolyl hydrazones.

**Figure 17.** Synthesis of morpholine-connected pyrazolidine.

66 Green Chemistry

**2.22. One-pot synthesis of** *o***-quinonic adducts**

**3. Conclusion**

**Acknowledgements**

**Figure 22.** Synthesis of *o*-quinonic adducts.

**Author details**

Saudi Arabia

cytotoxicity against MCF-7 and HEL tumoral cell lines (**Figure 22**) [22].

Dibenzo[a,h]anthracene derivatives were synthesized via a one-pot synthetic protocol with threecomponent reaction of 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes and 2-naphthol using InCl3 as catalyst under solvent-free condition. These *o*-quinonic adducts showed strong

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 69

The data of this chapter could be very helpful to identify the recently published approaches of

This book chapter was supported by a grant from the "Research Center of the Female Scientific

anticancer molecules synthesized via different green chemistry approaches.

and Medical Colleges," Deanship of Scientific Research, King Saud University.

Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh,

Shagufta Perveen\* and Areej Mohammad Al-Taweel

\*Address all correspondence to: shagufta792000@yahoo.com

#### **2.21. Solvent-free microwave-assisted synthesis of amide derivatives of ferulic acid**

In this research work, different amide derivatives of ferulic acid have been synthesized under solvent-free conditions by microwave-assisted reaction. These compounds were found to exhibit noticeable in vitro anticancer activity against breast (MDA-MB-231 and MCF-7), cervical (HeLa), lung (A549) and liver (HepG2) human cancer cell lines (**Figure 21**) [21].

**Figure 21.** Synthesis of ferulic acid derivatives.

#### **2.22. One-pot synthesis of** *o***-quinonic adducts**

Dibenzo[a,h]anthracene derivatives were synthesized via a one-pot synthetic protocol with threecomponent reaction of 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes and 2-naphthol using InCl3 as catalyst under solvent-free condition. These *o*-quinonic adducts showed strong cytotoxicity against MCF-7 and HEL tumoral cell lines (**Figure 22**) [22].

**Figure 22.** Synthesis of *o*-quinonic adducts.

### **3. Conclusion**

**2.21. Solvent-free microwave-assisted synthesis of amide derivatives of ferulic acid**

**Figure 20.** Synthesis of bacillamide analogues.

68 Green Chemistry

**Figure 21.** Synthesis of ferulic acid derivatives.

cal (HeLa), lung (A549) and liver (HepG2) human cancer cell lines (**Figure 21**) [21].

In this research work, different amide derivatives of ferulic acid have been synthesized under solvent-free conditions by microwave-assisted reaction. These compounds were found to exhibit noticeable in vitro anticancer activity against breast (MDA-MB-231 and MCF-7), cervi-

> The data of this chapter could be very helpful to identify the recently published approaches of anticancer molecules synthesized via different green chemistry approaches.

#### **Acknowledgements**

This book chapter was supported by a grant from the "Research Center of the Female Scientific and Medical Colleges," Deanship of Scientific Research, King Saud University.

#### **Author details**

Shagufta Perveen\* and Areej Mohammad Al-Taweel

\*Address all correspondence to: shagufta792000@yahoo.com

Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

## **References**

[1] Khaled AMA, Ghada HA, Abeer ME. Eco-friendly synthesis of novel cyanopyridine derivatives and their anticancer and PIM-1 kinase inhibitory activities. European Journal of Medicinal Chemistry 2017;**134**:357-365. DOI: 10.1016/j.ejmech.2017.04.024.

[11] Elgemeie GH, Farag AB, Amin KM, El-Badry OM and Hassan GS. Design, synthesis and cytotoxic evaluation of novel heterocyclic thioglycosides. Medicinal Chemistry 2014;**4**:814-

Green Chemistry and Synthesis of Anticancer Molecules http://dx.doi.org/10.5772/intechopen.70419 71

[12] Shailee VT, Julio AS, Vazquez-Tato MP, Aniket PS, Deepak KL, Anna PGN. Ultrasound mediated one-pot, three component synthesis, docking and ADME prediction of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo (3, 2-α) pyrimidine-6-carbonitrile derivatives as anticancer agents. Molecules 2016;**21**:894.

[13] Hue TBB, Quy TKH, Won KO, Duy DV, Yen NTC, Cuc TKT, Em CP, Phuong TT, Loan TT, Hieu VM. Microwave assisted synthesis and cytotoxic activity evaluations of new benzimidazole derivatives. Tetrahedron Letters 2016;**57**:887-891. DOI: 10.1016/j.tetlet.2016.01.042

[14] Abdel-Mohsen HT, Conrad J, Harms K, Nohr D, Beifuss U. Laccase-catalyzed green synthesis and cytotoxic activity of novel pyrimidobenzothiazoles and catechol thioethers.

[15] Adriana IG, Luiza G, Victor K, Luminita S, Thomas E, Valentin Z. Microwave-assisted synthesis of new selenazole derivatives with antiproliferative activity. Molecules 2013;**18**:

[16] Ahmed K, Rasala M, Nayak VL, Korrapati SB, Kumar GB, Anver BS, Jeevak SK, Abdullah A. Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4) inhibitors by catalyst-free, green approach. European Journal of Medicinal Chemistry

[17] Radhakrishnan SK, Meera M, Salem SA, Aseer M, Akbar I. Synthesis of new morpholine-connected pyrazolidine derivatives and their antimicrobial, antioxidant, and cytotoxic activities. Bioorganic & Medicinal Chemistry Letters 2017;**27**:66-71. DOI: 10.1016/j.

[18] Mahendra BB, Sambhaji TD, Amarsinh RD, Laxman UN, Vijay K, Dhiman S, Ramrao AM. New bithiazolyl hydrazones: Novel synthesis, characterization and antitubercular evaluation. Bioorganic & Medicinal Chemistry Letters 2017;**27**:288-294. DOI: 10.1016/j.

[19] Xiao-Hui X, Xiao-Wen G, Shi-Liang F, You-Zhen M, Shi-Wu C, Ling H. One-pot synthesis and biological evaluation of N-(aminosulfonyl)-4-podophyllotoxin carbamates as potential anticancer agents. Bioorganic & Medicinal Chemistry Letters 2017;**27**:2890-2894. DOI:

[20] Sunil K, Ranjana A, Virender K, Rachna S, Bhumi P, Pawan K, Dhirender K. Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-inflammatory agents. European

[21] Naresh K, Sandeep K, Sheenu A, Kumar N, Sham MS, Prasad VB, Partha R, Vikas P. Ferulic acid amide derivatives as anticancer and antioxidant agents: Synthesis, thermal,

Journal of Medicinal Chemistry 2016;**123**:718-726. DOI: 10.1016/j.ejmech.2016.07.033

RSC Advances 2017; **7**:17427-17441. DOI: 10.1039/c6ra28102h

4679-4688. DOI: 10.3390/molecules18044679

bmcl.2016.11.032

bmcl.2016.11.056

10.1016/j.bmcl.2017.04.082

2016;**108**:476-485. DOI: 10.1016/j.ejmech.2015.11.046

820. DOI: 10.4172/2161-0444.1000234

DOI: 10.3390/molecules21080894


[11] Elgemeie GH, Farag AB, Amin KM, El-Badry OM and Hassan GS. Design, synthesis and cytotoxic evaluation of novel heterocyclic thioglycosides. Medicinal Chemistry 2014;**4**:814- 820. DOI: 10.4172/2161-0444.1000234

**References**

70 Green Chemistry

[1] Khaled AMA, Ghada HA, Abeer ME. Eco-friendly synthesis of novel cyanopyridine derivatives and their anticancer and PIM-1 kinase inhibitory activities. European Journal

[2] Katarina MPG, Evgenija AD, Mihaly S, Janos G, Janos JC. Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives. Steroids 2015;**94**:31-40. DOI: 10.1016/j.steroids.2014.12.008 [3] Koduru SSP, Edupuganti VVSR. Design of new hybrid template by linking quinoline, triazole and dihydroquinoline pharmacophoric groups: A greener approach to novel polyazaheterocycles as cytotoxic agents. Bioorganic & Medicinal Chemistry Letters

[4] Srinivas NA, Mahendar P, Sadanandam A, Achaiah G, Malla RV. Synthesis, anticancer and MRP1 inhibitory activities of 4-alkyl/aryl-3,5-bis(carboethoxy/carbomethoxy)-1, 4-dihydro-2, 6-dimethylpyridines. Medicinal Chemistry Research 2013;**22**:147-155. DOI:

[5] Pinki Y, Kashmiri L, Ashwani K, Santosh KG, Sundeep J. Green synthesis and anticancer potential of chalcone linked-1,2,3-triazoles. European Journal of Medicinal Chemistry

[6] Chitrakar R, Arem Q, Darapaneni CM, Shashank KS. Design, synthesis and cytotoxicity studies of novel pyrazolo[1, 5-a] pyridine derivatives. European Journal of Medicinal

[7] Thangaraj A, Sadasivam M, Subashini G, Selvaraj S, Athar A, Palathurai SM. Biologically active perspective synthesis of heteroannulated 8-nitroquinolines with green chemistry approach. Bioorganic & Medicinal Chemistry Letters 2017;**27**:1538-1546. DOI: 10.1016/j.

[8] Assem B, Mohammad SI, Abdullah MA, Hazem AG, Sammer Y, Mahwish A, Nimra NS, Choudhary MI, Ruqaiya K, Zaheer U. Synthesis of pyrimidine-2,4,6-trione derivatives: Anti-oxidant, anti-cancer, a-glucosidase, b-glucuronidase inhibition and their molecular docking studies. Bioorganic Chemistry 2016;**68**:72-79. DOI: 10.1016/j.bioorg.2016.07.009

[9] Syed NAB, Ibrahim J, Vijay HM, Devidas TM, Muhammad S, Hassan MN, Muhammad WA. Synthesis of α, β-unsaturated carbonyl based compounds as acetylcholinesterase and butyrylcholinesterase inhibitors: Characterization, molecular modeling, QSAR studies and effect against amyloid β-induced cytotoxicity. European Journal of Medicinal

[10] Yuri FR, Cleiton MS, Daniel LS, Jeferson GS, Ana LTGR, Joao EC, Sergio AF, Angelo F. Phthalazine-triones: Calix[4]arene-assisted synthesis using green solvents and their anticancer activities against human cancer cells, Arabian Journal of Chemistry (2016) in

of Medicinal Chemistry 2017;**134**:357-365. DOI: 10.1016/j.ejmech.2017.04.024.

2015;**25**:1057-1063. DOI: 10.1016/j.bmcl.2015.01.012

2017;**126**:944-953. DOI: 10.1016/j.ejmech.2016.11.030

Chemistry 2017;**126**:277-285. DOI: 10.1016/j.ejmech.2016.11.037

Chemistry 2014;**83**:355-365. DOI: 10.1016/j.ejmech.2014.06.034

press. DOI: 10.1016/j.arabjc.2016.04.007

10.1007/s00044-012-9994-0

bmcl.2017.02.042


biological and computational studies. Medicinal Chemistry Research 2016;**25**:1175-1192. DOI: 10.1007/s00044-016-1562-6

**Chapter 5**

**Provisional chapter**

**The Role of Green Solvents and Catalysts at the Future**

Green chemistry is getting extended in many researches and industry areas. Not only pharmaceutical companies but also the other chemical industries started to take a step for green chemistry due to its advantages such as decreasing of waste and cost. With this respect, we have already witnessed that pharmaceutical companies searched out for green protocol when manufactured the pharmaceuticals. Green chemistry strategies can be seen in solvents, catalysts, and the others. So, we have briefly discussed the green solvents and nanocatalysts in this chapter. We hope that this chapter gives a brief consid-

**Keywords:** nanocatalyst, pharmaceutical company, green chemistry, environment

The demand of green chemistry for applying in the pharmaceutical and the other chemical industries is increasingly vital due to the fact that our world faces the environmental challenges of the twenty-first century. US Environmental Protection Agency (EPA) has suggested green chemistry for innovative technologies that reduce toxic, undesired waste, and environmental impact. Green chemistry is thus getting grew as an open light to afford a huge scientific area. After EPA, 12 principles of green chemistry have been gotten more attention and these principles have been considered more seriously by pharmaceutical companies since 1998. Pharmaceutical companies declared that they should improve the environmental performance by utilizing green chemistry. Not only pharmaceutical companies but also the other chemical industries started to take a step for green chemistry due to its advantages such as decreasing of waste and cost. It is assumed that green chemistry can save the industry an

**The Role of Green Solvents and Catalysts at the Future** 

DOI: 10.5772/intechopen.71018

© 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,

© 2018 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.

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

**of Drug Design and of Synthesis**

**of Drug Design and of Synthesis**

Additional information is available at the end of the chapter

eration of importance of green chemistry.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71018

Nurettin Menges

**Abstract**

**1. Introduction**

Nurettin Menges

[22] Idaira HF, Angel A, Patricia M, Matias L, Leandro F, Ana E. Indium catalyzed solventfree multicomponent synthesis of cytotoxic dibenzo[*a,h*]anthracenes from aldehydes, 2-hydroxy-1,4-naphthoquinone, and 2-naphthol. Tetrahedron 2014;**70**:8480-8487. DOI: 10.1016/j.tet.2014.09.076

**Provisional chapter**

## **The Role of Green Solvents and Catalysts at the Future of Drug Design and of Synthesis of Drug Design and of Synthesis**

**The Role of Green Solvents and Catalysts at the Future** 

DOI: 10.5772/intechopen.71018

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

biological and computational studies. Medicinal Chemistry Research 2016;**25**:1175-1192.

[22] Idaira HF, Angel A, Patricia M, Matias L, Leandro F, Ana E. Indium catalyzed solventfree multicomponent synthesis of cytotoxic dibenzo[*a,h*]anthracenes from aldehydes, 2-hydroxy-1,4-naphthoquinone, and 2-naphthol. Tetrahedron 2014;**70**:8480-8487. DOI:

DOI: 10.1007/s00044-016-1562-6

10.1016/j.tet.2014.09.076

72 Green Chemistry

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71018

#### **Abstract**

Green chemistry is getting extended in many researches and industry areas. Not only pharmaceutical companies but also the other chemical industries started to take a step for green chemistry due to its advantages such as decreasing of waste and cost. With this respect, we have already witnessed that pharmaceutical companies searched out for green protocol when manufactured the pharmaceuticals. Green chemistry strategies can be seen in solvents, catalysts, and the others. So, we have briefly discussed the green solvents and nanocatalysts in this chapter. We hope that this chapter gives a brief consideration of importance of green chemistry.

**Keywords:** nanocatalyst, pharmaceutical company, green chemistry, environment
