Colour-Based Natural Dyes and Pigments

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

Standards Overview - Bureau of Indian

[65] Samanta AK, Konar A, Agarwal P,

Standards. 2015;**28**(5):22-40

Datta S. Effects of chemical modifications of jute fabric with EDA and hydrazine on its textile related mechanical properties and dyeability. Journal of Polymer Materials.

[66] Samanta AK, Konar A, Chakraborti S, Datta S. Dyeing of jute fabric with tesu extract: Part-I: Effects of different mordants and dyeing process variables. Indian Journal of Fibre and Textile Research.

[67] Samanta AK, Konar A, Datta S. Dyeing of jute fabric with tesu extract: Part-II – Thermodynamic parameters

Journal of Fibre and Textile Research.

[68] Samanta AK, Deepali S, Mitu S. Application of single and mixture of selective natural dyes on cotton fabrics a scientific approach. Colourage.

[69] Shah HS, Gandhi RS. Instrumental Colour Measurements and Computer Aided Colour Matching for Textiles. Ahmedabad, India: Mahajan Book Distributers; 1990. pp. 76-84

Characteristics of fading of wool cloth dyed with selected natural dyestuffs on the basis of solar radiant energy. Dyes and Pigments. 2003;**58**(3):197-204

[70] Yoshizumi K, Crews PC.

and kinetics of dyeing. Indian

2010;**27**(3):203-236

2011;**36**(1):63-73

2012b;**37**(2):172-177

2003;**50**(10):29-42

[57] Shukla SR, Sundar PS. Anti-

Asian Dyer. 2008;**5**(2):37-39

2007;**32**(4):466-476

microbial and moth proofing of textiles.

[59] Samanta AK, Agarwal P. Application of mixtures of red sandal wood and other natural dyes for dyeing of jute fabric–studies on dye compatibility. International Dyer. 2008a;**193**(2):37-41

[60] Samanta AK, Agarwal P, Datta S. Dyeing of jute with binary mixtures of jackfruit wood and other natural dyes - study on colour performance and dye compatibility. Indian Journal

[61] Samanta AK, Agarwal P, Konar A,

[62] Samanta AK, Agarwal P, Singhee D, Datta S. Application of single and mixtures of red sandalwood and other natural dyes for dyeing of jute fabric: Studies on colour parameters/ colour fastness and compatibility. Journal of the Textile Institute.

[63] Samanta AK, Konar A. Technical Handbook on Natural Dye and Colouration. Kolkata, India: Department of Jute and Fibre

Technology, IJT (Calcutta University);

Standardization of methodologies for application of natural dyes on jute.

of Fibre and Textile Research.

Datta S. Characterization and application of purified selective dyes on jute. International Dyer.

2008b;**33**(2):171-180

2008c;**193**(3):25-32

2009;**100**(7):565-587

2012a. pp. 97-113

[64] Samanta AK, Konar A.

[58] Samanta AK, Agarwal P. Dyeing of jute and cotton fabrics using jackfruit wood extract: Part I-effects of mordanting and dyeing process variables on colour yield and colour fastness properties. Indian Journal of Fibre and Textile Research.

**170**

**Chapter 7**

**Abstract**

stability

**173**

**1. Introduction**

is a rich source of nitrate (NO3

represents the chemical structure of betanin.

Betanin: A Red-Violet

Pigment - Chemistry

and Applications

*Deepak Devadiga and T.N. Ahipa*

Nowadays, the demand for eco-friendly/nontoxic natural colorants is growing as an essential alternative to potentially harmful synthetic dyes. Betanin is the chief red pigment of beetroot, and it is the only betalain approved for use in food and pharmaceutical products as a natural red colorant. This chapter is mainly dealing with the betanin pigment, and also the chapter is subdivided into six sections covering the chemistry of betanin, extraction of color using various novel techniques (like microwave- and ultrasonic-assisted extraction) from raw plant material, biosynthesis of betanin followed by chemical synthesis of betanidin, and also the effect of pH, temperature, and light on the stability of betanin followed by its applications.

**Keywords:** beetroots, betanin, red pigment, extraction, chemical synthesis,

Vegetable beetroot (*Beta vulgaris* L.) has the notable scientific interest, because it

health effects through the endogen production of nitric oxide (NO) [1, 2]. There are two classes of pigment in plants, i.e., betalains and anthocyanins. Beetroots are the chief sources of betalains which is a water-soluble nitrogen pigment with heterocyclic ring, which can be further subdivided into two classes depending on chemical structure: betaxanthins comprising indicaxanthin; vulgaxanthin I and II, accountable for orange-yellow coloring; and betacyanins, such as betanin, isobetanin, neobetanin, and prebetanin, accountable for red-violet coloring [3, 4]. The most abundant betacyanin is betanin (betanidin 5-O-β-D-glucoside) and is the only pigment which is an approved natural colorant for the use in food products prescribed by the Food and Drug Administration (FDA) in the United States [5, 6]. **Figure 1**

According to experimental studies, raw beetroot generally contains water (87.1%), carbohydrate (7.6%), protein (1.7%), fat (0.1%), and betanin (0.03– 0.06%) [7]. In addition to natural food colorant property, betalain also exhibits antimicrobial, antiviral, and antioxidant activities [8]. Moreover, beetroot dye has nutrient value along with nontoxic nature; therefore, it even finds application in dyeing industry where the health aspect is a foremost criterion. Also, this natural dye extracted from beetroot is eco-friendly in nature and does not cause any environmental problems in contrast to the commercially available synthetic dyes [7].

), a compound with advantageous cardiovascular

## **Chapter 7**

## Betanin: A Red-Violet Pigment - Chemistry and Applications

*Deepak Devadiga and T.N. Ahipa*

## **Abstract**

Nowadays, the demand for eco-friendly/nontoxic natural colorants is growing as an essential alternative to potentially harmful synthetic dyes. Betanin is the chief red pigment of beetroot, and it is the only betalain approved for use in food and pharmaceutical products as a natural red colorant. This chapter is mainly dealing with the betanin pigment, and also the chapter is subdivided into six sections covering the chemistry of betanin, extraction of color using various novel techniques (like microwave- and ultrasonic-assisted extraction) from raw plant material, biosynthesis of betanin followed by chemical synthesis of betanidin, and also the effect of pH, temperature, and light on the stability of betanin followed by its applications.

**Keywords:** beetroots, betanin, red pigment, extraction, chemical synthesis, stability

## **1. Introduction**

Vegetable beetroot (*Beta vulgaris* L.) has the notable scientific interest, because it is a rich source of nitrate (NO3 ), a compound with advantageous cardiovascular health effects through the endogen production of nitric oxide (NO) [1, 2]. There are two classes of pigment in plants, i.e., betalains and anthocyanins. Beetroots are the chief sources of betalains which is a water-soluble nitrogen pigment with heterocyclic ring, which can be further subdivided into two classes depending on chemical structure: betaxanthins comprising indicaxanthin; vulgaxanthin I and II, accountable for orange-yellow coloring; and betacyanins, such as betanin, isobetanin, neobetanin, and prebetanin, accountable for red-violet coloring [3, 4]. The most abundant betacyanin is betanin (betanidin 5-O-β-D-glucoside) and is the only pigment which is an approved natural colorant for the use in food products prescribed by the Food and Drug Administration (FDA) in the United States [5, 6]. **Figure 1** represents the chemical structure of betanin.

According to experimental studies, raw beetroot generally contains water (87.1%), carbohydrate (7.6%), protein (1.7%), fat (0.1%), and betanin (0.03– 0.06%) [7]. In addition to natural food colorant property, betalain also exhibits antimicrobial, antiviral, and antioxidant activities [8]. Moreover, beetroot dye has nutrient value along with nontoxic nature; therefore, it even finds application in dyeing industry where the health aspect is a foremost criterion. Also, this natural dye extracted from beetroot is eco-friendly in nature and does not cause any environmental problems in contrast to the commercially available synthetic dyes [7].

**Figure 1.** *Chemical structure of betanin.*

## **2. Chemistry of betanin**

The first report on the crystallization of betanin was communicated by two independent groups Schmidt and Schonleben [9] and Wyler and Dreiding [10]. These two groups employed an electrophoretic strategy for betanin purification. Wyler and Dreiding [11] recognized three products which were formed by the alkaline degradation of betanidin (**Figure 2**); these were 4-methylpyridine-2,6 dicarboxylic acid, formic acid, and S-cyclodopa(5,6-dihydroxy-2,3-dihydroindole 2-carboxylic acid). When these three products are placed in correct relationship with one another, they form the betanidin's carbon skeleton and also the configuration of the second carbon [12].

## **2.1 Position of the β-D-glucosyl group**

To identify the position of β-D-glucosyl group, betanin was reacted with diazomethane in the presence of air yielded tetramethyl derivative of neobetanidin which was then converted to 6-methoxy-neobetanin-trimethylester by hydrolysis and acetylation [13, 14]. In the alkali condition, neobetanin cleaved to yield 5 hydroxy-6-methoxyindole 2-carboxylic acid. Its methyl ester was prepared from

acetylated molecule by degrading in basic condition, and further it was esterified and oxidized. Further, analysis of all these reactions together revealed the glucosyl residue position (**Figure 3**). In addition, NMR of betanin in trifluoroacetic acid and

The yellow-colored compound, i.e., 5,6-di-O-methylneobetanidin trimethyl ester, converted into colorless compound, i.e., 5,6-di-O-methyl-2,3-dehydro-11,12-dihydro-betanidin trimethyl ester, by the palladium-catalyzed disproportion-

hydrolytic studies of β-glucosidase [15] revealed that betanin was an O-β-D-

ation reaction which confirmed the existence of vinylene connecting group

Further, betanin can also be interconverted into betanidin. To prepare betanidin, betanin is initially reacted with the excess of L-proline in the presence of dilute ammonia which results in the formation of indicaxanthin which can later be

The drawbacks of conventional approaches, such as time-consuming methods, safety risks with some hazardous solvent systems, contaminated product, and comparatively less yields, have increased the demand for the novel processing methods

converted into betanidin by reacting it with excess S-cyclodopa [12].

(**Figure 4**) in the derivatives of neobetanidin [12].

*Reaction which confirmed the presence of vinylene connecting group.*

*Reactions that revealed the glucosyl residue position in betanin.*

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

glucopyranoside.

**Figure 3.**

**Figure 4.**

**3. Extraction techniques**

**175**

**Figure 2.** *Alkaline degradation of betanidin.*

*Betanin: A Red-Violet Pigment - Chemistry and Applications DOI: http://dx.doi.org/10.5772/intechopen.88939*

#### **Figure 3.**

**2. Chemistry of betanin**

*Chemical structure of betanin.*

**Figure 1.**

**Figure 2.**

**174**

*Alkaline degradation of betanidin.*

configuration of the second carbon [12].

**2.1 Position of the β-D-glucosyl group**

The first report on the crystallization of betanin was communicated by two independent groups Schmidt and Schonleben [9] and Wyler and Dreiding [10]. These two groups employed an electrophoretic strategy for betanin purification. Wyler and Dreiding [11] recognized three products which were formed by the alkaline degradation of betanidin (**Figure 2**); these were 4-methylpyridine-2,6 dicarboxylic acid, formic acid, and S-cyclodopa(5,6-dihydroxy-2,3-dihydroindole 2-carboxylic acid). When these three products are placed in correct relationship with one another, they form the betanidin's carbon skeleton and also the

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

To identify the position of β-D-glucosyl group, betanin was reacted with diazomethane in the presence of air yielded tetramethyl derivative of neobetanidin which was then converted to 6-methoxy-neobetanin-trimethylester by hydrolysis and acetylation [13, 14]. In the alkali condition, neobetanin cleaved to yield 5 hydroxy-6-methoxyindole 2-carboxylic acid. Its methyl ester was prepared from

*Reactions that revealed the glucosyl residue position in betanin.*

#### **Figure 4.**

*Reaction which confirmed the presence of vinylene connecting group.*

acetylated molecule by degrading in basic condition, and further it was esterified and oxidized. Further, analysis of all these reactions together revealed the glucosyl residue position (**Figure 3**). In addition, NMR of betanin in trifluoroacetic acid and hydrolytic studies of β-glucosidase [15] revealed that betanin was an O-β-Dglucopyranoside.

The yellow-colored compound, i.e., 5,6-di-O-methylneobetanidin trimethyl ester, converted into colorless compound, i.e., 5,6-di-O-methyl-2,3-dehydro-11,12-dihydro-betanidin trimethyl ester, by the palladium-catalyzed disproportionation reaction which confirmed the existence of vinylene connecting group (**Figure 4**) in the derivatives of neobetanidin [12].

Further, betanin can also be interconverted into betanidin. To prepare betanidin, betanin is initially reacted with the excess of L-proline in the presence of dilute ammonia which results in the formation of indicaxanthin which can later be converted into betanidin by reacting it with excess S-cyclodopa [12].

## **3. Extraction techniques**

The drawbacks of conventional approaches, such as time-consuming methods, safety risks with some hazardous solvent systems, contaminated product, and comparatively less yields, have increased the demand for the novel processing methods

### *Chemistry and Technology of Natural and Synthetic Dyes and Pigments*


#### **Table 1.**

*Betanin extraction from beetroot using different solvent systems.*

[16, 17]. To improve the betalain extraction, some of the pretreatment methods have been proposed, such as pulsed electric fields [18], gamma irradiation [8], and low-direct current electric fields [19]. But, such methods are quite expensive when compared to the solvent extraction techniques. Further, certain nonthermal techniques, like ultrasound (sonication) processing, and microwave-assisted extractions are also significantly productive in order to enhance the extraction yields of bioactive molecules with minimum degradation [17].

Neagu and Barbu [20] studied the betanin extraction from beetroot using different solvent systems by solid-liquid extraction technique (liquid/solid ratio is 5:1). **Table 1** presents the different extraction solvents used in this study. Results revealed that the highest betanin content of about 20 mg/g of beetroot was obtained with the use of weak acid solution (i.e., V8, using 0.5% citric acid +0.1% ascorbic acid). Also, they extracted the considerable amount of betanins by using ascorbic acid added solutions. Thus, it is clear that the acidic medium influences positively during the low-temperature extraction process.

In case of ultrasound (sonication) processing, ultrasonic-assisted extraction approach requires the use of ultrasound (with 20–2000 kHz frequencies range) which generally increases the cell wall permeability and generates the cavitations. Because of cavitations, cell membrane breaks down, and internal materials (color and oil) come out [21].

formed enzymatically over the shikimate pathway [24], which is the starting material for the biosynthesis of L-DOPA [25]. During this conversion, tyrosinase enzyme helped to convert tyrosine to DOPA through hydroxylation. Further, between carbons 4 and 5 of the L-DOPA cyclic ring was opened by 4,5-DOPA-extradiol dioxygenase enzyme to give 4,5-*seco*-DOPA [26–28] which is then converted into betalamic acid by intramolecular condensation between aldehyde and amine groups [25]. Further, L-DOPA is transformed into *o*-DOPAquinone in the presence of molecular oxygen [29]. Furthermore, it was spontaneously cyclized due to the nucleophilic attack of amino group on the ring system to yield *cyclo*-DOPA [23]. However, *cyclo*-DOPA can also be obtained from the cyclization of L-DOPA in the presence of

Finally, betanidin was formed by the formation of imine bond between *cyclo*-DOPA and betalamic acid which is then converted to betanin using betanidin-5-O-glucosyltransferase enzyme by connecting glucose unit of uridine diphosphateglucose (UDP-G) to the hydroxyl group in position 5 [31]. But this reaction can be reversed in the presence of β-glucosidase [32]. Additionally, it is also concluded that enzyme *cyclo*-DOPA-5-O-glucosyltransferase catalyzes the transport of glucose molecule on *cyclo*-DOPA, by which the *cyclo*-DOPA-glucoside condense with

cytochrome P450 (CYP76AD1) [30].

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

**Figure 5.**

**177**

*Biosynthesis of betanin.*

betalamic acid to yield the betanin [33].

On the other hand, microwave-assisted extractions are also contributed significantly to speed up the sample digestion and extraction of bioactive molecules from matrices. Here, the microwave energy has been utilized and employed in this extraction process. Moreover, this microwave energy induces molecular motion by the rotations of dipoles and migration of ions without varying the structure of the molecules provided the temperature of the system is not too high [21].

## **4. Biosynthesis of betanin**

The biosynthetic pathways for the betanin molecule are depicted in **Figure 5** [22]. Three enzymes such as 4,5-DOPA (dihydroxyphenylalanine)-extradiol-dioxygenase, tyrosinase, and betanidin-glucosyltransferase were involved in the biosynthesis of betalains in the cytoplasm [23]. From arogenic acid, the amino acid L-tyrosine was

*Betanin: A Red-Violet Pigment - Chemistry and Applications DOI: http://dx.doi.org/10.5772/intechopen.88939*

**Figure 5.** *Biosynthesis of betanin.*

[16, 17]. To improve the betalain extraction, some of the pretreatment methods have been proposed, such as pulsed electric fields [18], gamma irradiation [8], and low-direct current electric fields [19]. But, such methods are quite expensive when compared to the solvent extraction techniques. Further, certain nonthermal techniques, like ultrasound (sonication) processing, and microwave-assisted extractions are also significantly productive in order to enhance the extraction yields of bioac-

V8 0.5% citric acid + 0.1% ascorbic acid V9 0.2% citric acid + 0.1% ascorbic acid V10 20% ethanol + 1% citric acid V11 20% ethanol + 0.5% citric acid

**Variants Solvents** V1 Distilled water V2 1% citric acid V3 0.5% citric acid V4 0.2% citric acid V5 0.1% ascorbic acid V6 50% ethanol V7 20% ethanol

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

Neagu and Barbu [20] studied the betanin extraction from beetroot using different solvent systems by solid-liquid extraction technique (liquid/solid ratio is 5:1). **Table 1** presents the different extraction solvents used in this study. Results revealed that the highest betanin content of about 20 mg/g of beetroot was obtained with the use of weak acid solution (i.e., V8, using 0.5% citric acid +0.1% ascorbic acid). Also, they extracted the considerable amount of betanins by using ascorbic acid added solutions. Thus, it is clear that the acidic medium influences positively

In case of ultrasound (sonication) processing, ultrasonic-assisted extraction approach requires the use of ultrasound (with 20–2000 kHz frequencies range) which generally increases the cell wall permeability and generates the cavitations. Because of cavitations, cell membrane breaks down, and internal materials (color

On the other hand, microwave-assisted extractions are also contributed significantly to speed up the sample digestion and extraction of bioactive molecules from matrices. Here, the microwave energy has been utilized and employed in this extraction process. Moreover, this microwave energy induces molecular motion by the rotations of dipoles and migration of ions without varying the structure of the

The biosynthetic pathways for the betanin molecule are depicted in **Figure 5** [22]. Three enzymes such as 4,5-DOPA (dihydroxyphenylalanine)-extradiol-dioxygenase, tyrosinase, and betanidin-glucosyltransferase were involved in the biosynthesis of betalains in the cytoplasm [23]. From arogenic acid, the amino acid L-tyrosine was

molecules provided the temperature of the system is not too high [21].

tive molecules with minimum degradation [17].

*Betanin extraction from beetroot using different solvent systems.*

during the low-temperature extraction process.

and oil) come out [21].

**Table 1.**

**4. Biosynthesis of betanin**

**176**

formed enzymatically over the shikimate pathway [24], which is the starting material for the biosynthesis of L-DOPA [25]. During this conversion, tyrosinase enzyme helped to convert tyrosine to DOPA through hydroxylation. Further, between carbons 4 and 5 of the L-DOPA cyclic ring was opened by 4,5-DOPA-extradiol dioxygenase enzyme to give 4,5-*seco*-DOPA [26–28] which is then converted into betalamic acid by intramolecular condensation between aldehyde and amine groups [25]. Further, L-DOPA is transformed into *o*-DOPAquinone in the presence of molecular oxygen [29]. Furthermore, it was spontaneously cyclized due to the nucleophilic attack of amino group on the ring system to yield *cyclo*-DOPA [23]. However, *cyclo*-DOPA can also be obtained from the cyclization of L-DOPA in the presence of cytochrome P450 (CYP76AD1) [30].

Finally, betanidin was formed by the formation of imine bond between *cyclo*-DOPA and betalamic acid which is then converted to betanin using betanidin-5-O-glucosyltransferase enzyme by connecting glucose unit of uridine diphosphateglucose (UDP-G) to the hydroxyl group in position 5 [31]. But this reaction can be reversed in the presence of β-glucosidase [32]. Additionally, it is also concluded that enzyme *cyclo*-DOPA-5-O-glucosyltransferase catalyzes the transport of glucose molecule on *cyclo*-DOPA, by which the *cyclo*-DOPA-glucoside condense with betalamic acid to yield the betanin [33].

## **5. Chemical synthesis of betanidin**

The chemical synthesis of betanidin is illustrated in **Figure 6** [34]. For the synthesis of betanidin, 4-hydroxypyridine-2,6-dicarboxylic acid is used as the starting material which upon hydrogenation and followed by esterification yields all products in *cis* form. Further, Pfitzner-Moffatt oxidation reaction converted the secondary hydroxyl group to a ketone. In the next step, it is converted to semicarbazide using Horner-Wittig reagent. Then, the obtained semicarbazide is further hydrolyzed to give unsaturated ketone, which is converted to betalamic acid by Pfitzner-Moffatt oxidation. Reacting betalamic acid with L-cyclo-DOPA methyl ester yielded betanidin trimethyl ester which is then converted to betanidin through acid hydrolysis by using concentrated hydrochloric acid.

around 530 nm in the visible range for the red beet juice which was attributed to the betanin pigment. As the solvent changed from ethanol (532 nm) to water (542 nm) and to methanol (544 nm), absorption maximum shifted towards longer wavelengths. These results showed that methanolic and aqueous extracts mainly contain

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

*The UV-vis spectra of beetroot extracts in different solvents (ethanol, methanol, and water).*

**Figure 7.**

**Figure 8.**

**179**

*FT-IR spectrum of betanin (scale range, 400–4000 cm<sup>1</sup>*

*).*

**Figure 6.** *Chemical synthesis of betanidin.*

## **6. Characterization of betanin**

## **6.1 UV-vis absorption spectra of beetroot extracts**

The UV-vis spectra of beetroot extracts in different solvents (ethanol, methanol, and water) are depicted in **Figure 7** [35]. Strong absorption band was observed at

**5. Chemical synthesis of betanidin**

**6. Characterization of betanin**

*Chemical synthesis of betanidin.*

**Figure 6.**

**178**

**6.1 UV-vis absorption spectra of beetroot extracts**

The UV-vis spectra of beetroot extracts in different solvents (ethanol, methanol, and water) are depicted in **Figure 7** [35]. Strong absorption band was observed at

The chemical synthesis of betanidin is illustrated in **Figure 6** [34]. For the synthesis of betanidin, 4-hydroxypyridine-2,6-dicarboxylic acid is used as the starting material which upon hydrogenation and followed by esterification yields all products in *cis* form. Further, Pfitzner-Moffatt oxidation reaction converted the secondary hydroxyl group to a ketone. In the next step, it is converted to

semicarbazide using Horner-Wittig reagent. Then, the obtained semicarbazide is further hydrolyzed to give unsaturated ketone, which is converted to betalamic acid by Pfitzner-Moffatt oxidation. Reacting betalamic acid with L-cyclo-DOPA methyl ester yielded betanidin trimethyl ester which is then converted to betanidin through

acid hydrolysis by using concentrated hydrochloric acid.

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

around 530 nm in the visible range for the red beet juice which was attributed to the betanin pigment. As the solvent changed from ethanol (532 nm) to water (542 nm) and to methanol (544 nm), absorption maximum shifted towards longer wavelengths. These results showed that methanolic and aqueous extracts mainly contain

**Figure 7.** *The UV-vis spectra of beetroot extracts in different solvents (ethanol, methanol, and water).*

**Figure 8.** *FT-IR spectrum of betanin (scale range, 400–4000 cm<sup>1</sup> ).*

betanidin, whereas ethanolic extract mainly contains betanin. The intensity of absorption maxima for the aqueous and methanolic extracts is approximately equal, higher than the ethanolic extract.

Further, partially overlapped two absorption bands were observed in case of aqueous and methanolic extracts. For aqueous extract the second band has an absorption maximum at 515 nm and for aqueous extract at 509 nm. These bands are only observed in high concentrated extract, and it vanishes as the solution diluted. Hence, it can be attributed to the formation of supramolecular structure in the concentrated solutions.

## **6.2 FT-IR data of betanin**

Different characteristic absorption bands corresponding to the functional groups of betanins were observed, and its FT-IR spectrum is shown in **Figure 8** [36]. The absorption band around 3359 cm<sup>1</sup> was ascribed to the ▬OH bond stretching vibration [37]; on the other hand, the absorption band around 1624 cm<sup>1</sup> was ascribed to the C〓N bond stretching vibration [38, 39]. The next absorption band located at 1378 cm<sup>1</sup> was ascribed to the C▬H bond extension stretching vibration, while the absorption band at 1243 cm<sup>1</sup> was ascribed to the C▬O bond of the carboxylic acid stretching vibration [38, 39]. Another absorption band centered at 1073 cm<sup>1</sup> was ascribed to the C▬O▬C linked symmetric stretching vibration [40], the absorption band at 945 cm<sup>1</sup> was ascribed to the C▬H bond deformation, and lastly the absorption band at 879 cm<sup>1</sup> was ascribed to the C▬COOH bond stretching vibrations [41].

#### **6.3 <sup>1</sup> H, 13C, and LC-1 H NMR data of betanin**

The <sup>1</sup> H and 13C NMR data of betanin was obtained by dissolving it in D2O, and its LC-<sup>1</sup> H NMR data was also obtained by dissolving it in acetonitrile (MeCN)/D2O/ 0.05% trifluoroacetic acid (TFA) using 500 MHz frequency at 25°C. **Figure 9** represents the LC-1 H NMR spectrum of betanin and followed by **Table 2** which represents the <sup>1</sup> H, 13C, and LC-1 H NMR data of betanin [42].

**6.4 Mass spectrum of betanin**

*Chemical shifts were not observable.*

*bt = broad triplet, bm = broad multiplet.*

a/b 3.85, *dd*, 1.6, 12.3

60

**Table 2.** *1*

*H, 13C, and LC-1*

*a*

*b*

**181**

**Atom numbering <sup>1</sup>**

3a/b 3.53, *dd*, 11.5, 16.9

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

14a/b 3.20, *bm*

**H NMR δ-[ppm], mult, J [Hz]**

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

3.10, *dd*, 4.3, 16.8

3.12, *bm*

3.70, *dd*, 5.3, 12.3

*H NMR data of betanin.*

**13C NMR δ-[ppm], mult, J [Hz] LC-<sup>1</sup>**

2 4.92, *dd*, 3.1, 10.3 65.0 5.23, *dd*, 2.0, 10.2

 4.40, *bt*, 7.1 53.1 Overlapped by D2O 17 — —<sup>a</sup> — 6.22, *bs* —<sup>a</sup> 6.34, *bs* 19 — —<sup>a</sup> — 20 — —<sup>a</sup> — <sup>0</sup> 4.98, *d*, 7.4 101.4 5.00, *d*, 6.9 <sup>0</sup> 3.55 (overlap) 75.7 3.41–3.59 (overlap) <sup>0</sup> 3.55 (overlap) 73.9 3.41–3.59 (overlap) <sup>0</sup> 3.41 (overlap) 69.3 3.41–3.59 (overlap) <sup>0</sup> 3.52 (overlap) 76.2 3.41–3.59 (overlap)

 7.06, *s* 113.9 7.13, *s* — 144.0 — — 146.1 — 6.98, *bs* 100.0 7.12, *bs* — 137.4 — — 124.1 — — 175.8 — 8.19, *bs* (*d*, 12.6)<sup>b</sup> 144.4 8.38, *bd*, ≈11 5.84, *bs* (*d*, 12.6)<sup>b</sup> 106.9 6.04, *bd*, ≈11 13 — —<sup>a</sup> —

**H NMR δ-[ppm], mult, J [Hz]**

3.31, *dd*, 2.0, 16.7

3.41–3.59 (overlap)

3.70, *dd*, 5.5, 12.6

32.7 3.66, *dd*, 10.4, 16.7

26.5 3.21, *dd*, 7.4, 17.2

60.6 3.86, *dd*, 1.4, 12.6

molecular ion (*m*/*z* 551, [M+H]<sup>+</sup>

spectrum of betanin molecule [43].

**6.5 Thermogravimetric (TG) analysis**

The mass spectrometry of betanin in the positive ionization mode exhibited a

*After acidification (TFA) to pH 2, s = singlet, d = doublet, dd = doublet of doublet, bs = broad singlet, bd = broad doublet,*

The dynamic thermogravimetric (TG) analysis was conducted on fresh betanin and dried betanin (**Figure 11a** and **b**) [44]. Since fresh betanin contains water, immediate mass loss is noted in the temperature range of 40–100°C. Further, the degradation temperature of betanin dye is noted at the temperature of about 204°C.

, 100%). **Figure 10** represents the obtained mass

**Figure 9.** *LC-1 H NMR spectrum of betanin.*


#### *Betanin: A Red-Violet Pigment - Chemistry and Applications DOI: http://dx.doi.org/10.5772/intechopen.88939*

*a Chemical shifts were not observable.*

*b After acidification (TFA) to pH 2, s = singlet, d = doublet, dd = doublet of doublet, bs = broad singlet, bd = broad doublet, bt = broad triplet, bm = broad multiplet.*

**Table 2.**

betanidin, whereas ethanolic extract mainly contains betanin. The intensity of absorption maxima for the aqueous and methanolic extracts is approximately equal,

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

Further, partially overlapped two absorption bands were observed in case of aqueous and methanolic extracts. For aqueous extract the second band has an absorption maximum at 515 nm and for aqueous extract at 509 nm. These bands are only observed in high concentrated extract, and it vanishes as the solution diluted. Hence, it can be attributed to the formation of supramolecular structure in the

Different characteristic absorption bands corresponding to the functional groups of betanins were observed, and its FT-IR spectrum is shown in **Figure 8** [36]. The absorption band around 3359 cm<sup>1</sup> was ascribed to the ▬OH bond stretching vibration [37]; on the other hand, the absorption band around 1624 cm<sup>1</sup> was ascribed to the C〓N bond stretching vibration [38, 39]. The next absorption band located at 1378 cm<sup>1</sup> was ascribed to the C▬H bond extension stretching vibration, while the absorption band at 1243 cm<sup>1</sup> was ascribed to the C▬O bond of the carboxylic acid stretching vibration [38, 39]. Another absorption band centered at 1073 cm<sup>1</sup> was ascribed to the C▬O▬C linked symmetric stretching vibration [40], the absorption band at 945 cm<sup>1</sup> was ascribed to the C▬H bond deformation, and lastly the absorption band at 879 cm<sup>1</sup> was ascribed to the C▬COOH bond

H and 13C NMR data of betanin was obtained by dissolving it in D2O, and

H NMR data was also obtained by dissolving it in acetonitrile (MeCN)/D2O/

H NMR spectrum of betanin and followed by **Table 2** which repre-

0.05% trifluoroacetic acid (TFA) using 500 MHz frequency at 25°C. **Figure 9** rep-

H NMR data of betanin [42].

**H NMR data of betanin**

higher than the ethanolic extract.

concentrated solutions.

**6.2 FT-IR data of betanin**

stretching vibrations [41].

**H, 13C, and LC-1**

H, 13C, and LC-1

**6.3 <sup>1</sup>**

The <sup>1</sup>

resents the LC-1

its LC-<sup>1</sup>

sents the <sup>1</sup>

**Figure 9.** *LC-1*

**180**

*H NMR spectrum of betanin.*

*1 H, 13C, and LC-1 H NMR data of betanin.*

## **6.4 Mass spectrum of betanin**

The mass spectrometry of betanin in the positive ionization mode exhibited a molecular ion (*m*/*z* 551, [M+H]<sup>+</sup> , 100%). **Figure 10** represents the obtained mass spectrum of betanin molecule [43].

## **6.5 Thermogravimetric (TG) analysis**

The dynamic thermogravimetric (TG) analysis was conducted on fresh betanin and dried betanin (**Figure 11a** and **b**) [44]. Since fresh betanin contains water, immediate mass loss is noted in the temperature range of 40–100°C. Further, the degradation temperature of betanin dye is noted at the temperature of about 204°C.

and pH 7. Further, the betanin compound is more stable between pH 4 and 5. However, betanin in beet juice is far more stable at pH 5, which reveals a protective effect by the constituents of juice. The rates of degradation for

**Figure 12.**

**Figure 13.**

**183**

*Rates of degradation for betanin molecule in a system at 100°C at pH 3, 5, and 7.*

*Visible spectra of betanin compound at pH 2, 5, and 9.*

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

**Figure 10.** *Mass spectrum of betanin.*

**Figure 11.** *(a) TG curves and (b) derivative TG curves of fresh and dried betanin.*

Furthermore, the dried sample has also exhibited similar behavior except the mass loss at the beginning (in the temperature range of 40–100°C).

## **7. Factors effecting the stability of betanin**


and pH 7. Further, the betanin compound is more stable between pH 4 and 5. However, betanin in beet juice is far more stable at pH 5, which reveals a protective effect by the constituents of juice. The rates of degradation for

**Figure 12.** *Visible spectra of betanin compound at pH 2, 5, and 9.*

**Figure 13.** *Rates of degradation for betanin molecule in a system at 100°C at pH 3, 5, and 7.*

Furthermore, the dried sample has also exhibited similar behavior except the mass

• *pH*: in the buffers of pH 2–9, betanin was stored at 4°C for 7 days and measured the visible spectra at both starting and end of this time span. No shifts in the absorption maxima were noted in between pH 4 and pH 7. A shift of about 2 nm

towards a shorter wavelength with a decreased absorbance intensity was observed in case of the buffer which has pH less than 4. In the 575–650 nm region, the spectrum has slightly increased absorbance, and the solution color changed to red-violet from red. While in case of the solutions which has pH value of above 7, i.e., at pH 9, the absorption maximum moved to a longer wavelength region (544 nm) by decreasing the intensity. In the 575–650 nm and 400–450 nm wavelength regions, the absorbance increased to a considerable extent, and the solution color changed to violet from red. These results showed that between pH 3 and pH 7, storage had no effect on betanin solutions, and above and below of these pH values causes the considerable losses of betanin. Visible spectra of betanin compound at pH 2, 5, and 9 are illustrated in **Figure 12** [12, 45].

• *Temperature*: on heating the red color of betanin solutions starts to diminish, and finally it turns to brown color. The color loss was followed by the betanin assay, and the rate indicates that it follows first-order kinetics. The graph indicates that at 100°C, the degradation rate at pH 5 is still less than it is at pH 3

loss at the beginning (in the temperature range of 40–100°C).

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

**7. Factors effecting the stability of betanin**

*(a) TG curves and (b) derivative TG curves of fresh and dried betanin.*

**Figure 11.**

**182**

**Figure 10.**

*Mass spectrum of betanin.*

betanin molecule in a system at 100°C at pH 3, 5, and 7 are depicted in **Figure 13** [12, 45].

• *Light*: at 15°C and pH 7, the presence of light increased the rate of degradation by 15.6% and air (rather than nitrogen) by 14.6%. Both light and air together increased the rate by 28.6% [12, 42].

*8.1.3 Effect of temperature on dyeing*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*8.1.4 Effect of time span on dyeing*

**Fastness to dry rubbing**

**Fastness to wet rubbing**

None 5\* 5\* 4\*–5\* 4\*–5\* 3\* Manganese sulfate 5\* 5\* 4\* 4\* 4\* Ferrous sulfate 4\* 4\* 3\* 3\* 3\* Zinc sulfate 5\* 5\* 4\*–5\* 4\*–5\* 4\*

Cobalt sulfate 5\* 5\* 4\*–5\* 4\*–5\* 5\*

*The fastness properties of betanin-dyed acrylic fabrics represented with the rating scale using star (\*).*

**Fastness to washing**

4\* 4\* 3\* 3\* 3\*

**Fastness to water**

**Fastness to light**

time increases [43].

*8.1.5 Color fastness*

Aluminum potassium sulfate

**Table 3.**

**185**

As the temperature of dyeing increases, the color strength increases up to 50°C, and further by increasing the dyeing temperature, the color strength decreased slowly which is attributed to the decrease in stability of dye at higher temperatures [43].

Color strength increases as the dyeing time increases up to 30 min; from 30 to 45 min, there is no change in color strength, and then it started to decrease as the

The fastness properties of betanin-dyed acrylic fabrics are shown in **Table 3**. The fastness properties of the dyed samples were examined according to ISO standard methods, the specific tests conducted for color fastness to rubbing is as per ISO 105-X12:1987, the color fastness to water is as per ISO 105-E01:1989, the color fastness to washing is as per ISO 105-C02:1989, and the color fastness to light is as per ISO 105-B02:1988 (carbon arc) [43]. It was noted that rubbing, washing, and water fastness of unmordanted acrylic fabrics exhibited significantly good property. But, the light fastness of unmordanted acrylic fabrics was found to be bad. However, light fastness was found to increase from rating 3\* to 4\* in premordanted fabrics using manganese sulfate and ferrous sulfate, and light fastness was increased from rating 3\* to 5\* by using cobalt sulfate. Nevertheless, the other mordants did not affect the light fastness of premordanted fabrics. It was found that for the improvements of color strength and light fastness, cobalt sulfate was established as the best mordant [43]. Similarly, Guesmi et al. [46] in the year 2013 studied the dyeing of wool fabric using betanin and chlorophyll-a as biomordant. In a dye bath having sodium chloride (0–5 g/L) and a dye with 40:1 liquor ratio, wool fabric was dyed using conventional heating. Results revealed that the increase in the concentration of biomordant increases the color strength values. They also investigated the effect of variables on the color of dyed fibers and noted that from pH 3.5 to pH 4.5, the color strength considerably increases, the color strength was found to be better without salt than with salt, and the color strength of dyed wool increases as the increase in temperature was up to 40°C and starts to decrease slowly till 50°C. Further increase in temperature, the color strength decreases in pronounced manner. According to the authors, color strength increases with the time span (up to 45 min) of dyeing, and

## **8. Applicability of betanin**

#### **8.1 Dyeing acrylic fabric**

Pure betanin dye can compete with synthetic dyes in color depth shade properties and in color fastness properties. Guesmi et al. [43] studied the dyeing of betanin on modified acrylic fabrics and evaluated the effect of dye bath pH, salt concentration, dyeing time, and temperature on dyeing. In a dye bath having sodium chloride (0–15 g/L) and a dye of 30 mg/L concentration with the 40:1 liquor ratio, modified acrylic fabric was dyed using conventional heating.

#### *8.1.1 Effect of pH on dyeing*

Over the pH range 1–5, increase in pH increases the adsorption of betanin onto acrylic fabric. Color strength decreases as the pH increased above 5. Generally, amino functional groups of acrylic fibers get protonated as the pH value decreases. Thus, ion-ion forces induced with ionized carboxyl groups in betanin. Betanin may exist in cationized or on monoanion form in a strongly acidic environment which results in the lower depth of dyeing at pH less than 4, and also it is due to the betanin stability loss at low pH [43].

At pH 5 maximal color strength was observed, whereas at pH 4, little decrease in color strength was observed; this is attributed to the increased carboxyl groups in this range and to the high thermal stability of betanin molecule. The number of protonated terminal amino functional groups of fabric decreases at pH > 5, which causes the decreased ionic interaction between the carboxylate anion of the dye and acrylic fibers, thus lowering its dye ability. The structures of betanin molecule as a pH varied are depicted in **Figure 14** [43].

### *8.1.2 Effect of salt addition*

Color strength decreases as the salt concentration increases, hence dyeing without salt addition is the best condition [43].

**Figure 14.** *Structures of betanin molecule as a pH varied [46].*

## *8.1.3 Effect of temperature on dyeing*

As the temperature of dyeing increases, the color strength increases up to 50°C, and further by increasing the dyeing temperature, the color strength decreased slowly which is attributed to the decrease in stability of dye at higher temperatures [43].

## *8.1.4 Effect of time span on dyeing*

Color strength increases as the dyeing time increases up to 30 min; from 30 to 45 min, there is no change in color strength, and then it started to decrease as the time increases [43].

## *8.1.5 Color fastness*

betanin molecule in a system at 100°C at pH 3, 5, and 7 are depicted in

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

• *Light*: at 15°C and pH 7, the presence of light increased the rate of degradation by 15.6% and air (rather than nitrogen) by 14.6%. Both light and air together

Pure betanin dye can compete with synthetic dyes in color depth shade properties and in color fastness properties. Guesmi et al. [43] studied the dyeing of betanin on modified acrylic fabrics and evaluated the effect of dye bath pH, salt concentration, dyeing time, and temperature on dyeing. In a dye bath having sodium chloride (0–15 g/L) and a dye of 30 mg/L concentration with the 40:1 liquor ratio, modified

Over the pH range 1–5, increase in pH increases the adsorption of betanin onto acrylic fabric. Color strength decreases as the pH increased above 5. Generally, amino functional groups of acrylic fibers get protonated as the pH value decreases. Thus, ion-ion forces induced with ionized carboxyl groups in betanin. Betanin may exist in cationized or on monoanion form in a strongly acidic environment which results in the lower depth of dyeing at pH less than 4, and also it is due to the

At pH 5 maximal color strength was observed, whereas at pH 4, little decrease in color strength was observed; this is attributed to the increased carboxyl groups in this range and to the high thermal stability of betanin molecule. The number of protonated terminal amino functional groups of fabric decreases at pH > 5, which causes the decreased ionic interaction between the carboxylate anion of the dye and acrylic fibers, thus lowering its dye ability. The structures of betanin molecule as a

Color strength decreases as the salt concentration increases, hence dyeing with-

**Figure 13** [12, 45].

**8. Applicability of betanin**

**8.1 Dyeing acrylic fabric**

*8.1.1 Effect of pH on dyeing*

betanin stability loss at low pH [43].

pH varied are depicted in **Figure 14** [43].

out salt addition is the best condition [43].

*Structures of betanin molecule as a pH varied [46].*

*8.1.2 Effect of salt addition*

**Figure 14.**

**184**

increased the rate by 28.6% [12, 42].

acrylic fabric was dyed using conventional heating.

The fastness properties of betanin-dyed acrylic fabrics are shown in **Table 3**. The fastness properties of the dyed samples were examined according to ISO standard methods, the specific tests conducted for color fastness to rubbing is as per ISO 105-X12:1987, the color fastness to water is as per ISO 105-E01:1989, the color fastness to washing is as per ISO 105-C02:1989, and the color fastness to light is as per ISO 105-B02:1988 (carbon arc) [43]. It was noted that rubbing, washing, and water fastness of unmordanted acrylic fabrics exhibited significantly good property. But, the light fastness of unmordanted acrylic fabrics was found to be bad. However, light fastness was found to increase from rating 3\* to 4\* in premordanted fabrics using manganese sulfate and ferrous sulfate, and light fastness was increased from rating 3\* to 5\* by using cobalt sulfate. Nevertheless, the other mordants did not affect the light fastness of premordanted fabrics. It was found that for the improvements of color strength and light fastness, cobalt sulfate was established as the best mordant [43].

Similarly, Guesmi et al. [46] in the year 2013 studied the dyeing of wool fabric using betanin and chlorophyll-a as biomordant. In a dye bath having sodium chloride (0–5 g/L) and a dye with 40:1 liquor ratio, wool fabric was dyed using conventional heating. Results revealed that the increase in the concentration of biomordant increases the color strength values. They also investigated the effect of variables on the color of dyed fibers and noted that from pH 3.5 to pH 4.5, the color strength considerably increases, the color strength was found to be better without salt than with salt, and the color strength of dyed wool increases as the increase in temperature was up to 40°C and starts to decrease slowly till 50°C. Further increase in temperature, the color strength decreases in pronounced manner. According to the authors, color strength increases with the time span (up to 45 min) of dyeing, and


### **Table 3.**

*The fastness properties of betanin-dyed acrylic fabrics represented with the rating scale using star (\*).*

then it starts to decrease because betanin losses thermal stability, and also it starts to escape from the fiber. Dye exhaustion was examined in both ultrasonic and conventional dyeing approaches. It was exhibited that in a shorter time span of dyeing, sonication increases the dye exhaustion from rating of 30% to rating of 60%. The fastness properties of dyed wool were studied against wet rubbing, light, washing, and dry rubbing. Unmordanted and mordanted samples have good fastness properties too.

## **8.2 Medicinal application**

Antioxidant activity of betanin in biological lipid domain has been exhibited in human macromolecules, like lipoproteins of low density, whole cells, and membranes [2]. Moreover, betanin has attracted researchers because of its antiinflammatory activities and hepatic safety activities in whole human cells [47]. In cultured endothelia cells, this molecule regulates the redox-mediated signal transduction pathways which is required in responses during inflammation, and betanin also showed antiproliferative effects on tumor cell lines in human [48–50]. In both tumoral and healthy hepatic cell lines in the human body, betanin translocates the antioxidant response element (erythroid 2-related factor 2 (Nrf2)) from the place of cytosol to the place of nuclear domain, which regulates m-RNA and protein levels of antioxidant/detoxifying enzymes, which includes heme oxygenase-1, NAD(P)H quinone dehydrogenase-1, and glutathione S-transferase and, in these cells, bears anticarcinogenic and hepatoprotective effects [51]. Also, it exhibits antidiabetes properties by controlling the activities of liver markers enzymes [52–54].

## **8.3 Betanin as food colorant**

Betanin is the oldest and most abundant red food colorant which has been established in the market, which is noted as E-162 in the European Union and in the United Sates; it is known as 73.40 in the twenty-first chapter of the Code of Federal Regulations (CFR) section of the Food and Drug Earth Administration [2, 5, 6].

Betanins are most commonly used for coloring of ice cream and powdered soft drink beverages. Additionally, betanin is used in some of the sugar confectionery, like sugar coatings, fruit or ice cream fillings, fondants, and sugar strands. At the final part of the processing, it can be added while preparing hot processed candies. Also, it is used in soups as well as bacon and tomato.

## **9. Summary**

In this chapter, the first and second section covered the chemistry of betanin which contains reactions that revealed the glucosyl residue position and presence of vinylene connecting group in betanin. Further, the third section narrated the extraction techniques which mainly included the microwave- and ultrasonicassisted extraction method. Furthermore, the fourth and fifth sections elucidated the biosynthesis of betanin molecule and chemical synthesis of betanidin molecule, respectively. In addition, different characterization techniques were also explicated in the sixth section which includes UV-Vis, FT-IR, <sup>1</sup> H NMR, 13C NMR, LC-1 H NMR, mass spectrum, and thermogravimetric analysis of betanin. Also, the factors effecting the stability of betanin were explained in the seventh section which covers the effect of pH, temperature, and light on the stability of betanin. Lastly, the applicability of betanin was taken into account in the eighth section which comprised of dyeing of acrylic fabric, dyeing of wool fabric, and medicinal and food colorant applications of betanin.

**Author details**

**187**

Deepak Devadiga and T.N. Ahipa\*

provided the original work is properly cited.

Centre for Nano and Material Sciences, Jain University, Bangalore, India

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: tn.ahipa@jainuniversity.ac.in

*Betanin: A Red-Violet Pigment - Chemistry and Applications*

*DOI: http://dx.doi.org/10.5772/intechopen.88939*

*Betanin: A Red-Violet Pigment - Chemistry and Applications DOI: http://dx.doi.org/10.5772/intechopen.88939*

then it starts to decrease because betanin losses thermal stability, and also it starts to escape from the fiber. Dye exhaustion was examined in both ultrasonic and conventional dyeing approaches. It was exhibited that in a shorter time span of dyeing, sonication increases the dye exhaustion from rating of 30% to rating of 60%. The fastness properties of dyed wool were studied against wet rubbing, light, washing, and dry rubbing. Unmordanted and mordanted samples have good fastness properties too.

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

Antioxidant activity of betanin in biological lipid domain has been exhibited in human macromolecules, like lipoproteins of low density, whole cells, and membranes [2]. Moreover, betanin has attracted researchers because of its antiinflammatory activities and hepatic safety activities in whole human cells [47]. In cultured endothelia cells, this molecule regulates the redox-mediated signal transduction pathways which is required in responses during inflammation, and betanin also showed antiproliferative effects on tumor cell lines in human [48–50]. In both tumoral and healthy hepatic cell lines in the human body, betanin translocates the antioxidant response element (erythroid 2-related factor 2 (Nrf2)) from the place of cytosol to the place of nuclear domain, which regulates m-RNA and protein levels of antioxidant/detoxifying enzymes, which includes heme oxygenase-1, NAD(P)H quinone dehydrogenase-1, and glutathione S-transferase and, in these cells, bears anticarcinogenic and hepatoprotective effects [51]. Also, it exhibits antidiabetes properties by controlling the activities of liver markers enzymes [52–54].

Betanin is the oldest and most abundant red food colorant which has been established in the market, which is noted as E-162 in the European Union and in the United Sates; it is known as 73.40 in the twenty-first chapter of the Code of Federal Regulations (CFR) section of the Food and Drug Earth Administration [2, 5, 6]. Betanins are most commonly used for coloring of ice cream and powdered soft drink beverages. Additionally, betanin is used in some of the sugar confectionery, like sugar coatings, fruit or ice cream fillings, fondants, and sugar strands. At the final part of the processing, it can be added while preparing hot processed candies.

In this chapter, the first and second section covered the chemistry of betanin which contains reactions that revealed the glucosyl residue position and presence of vinylene connecting group in betanin. Further, the third section narrated the extraction techniques which mainly included the microwave- and ultrasonicassisted extraction method. Furthermore, the fourth and fifth sections elucidated the biosynthesis of betanin molecule and chemical synthesis of betanidin molecule, respectively. In addition, different characterization techniques were also explicated

mass spectrum, and thermogravimetric analysis of betanin. Also, the factors effecting the stability of betanin were explained in the seventh section which covers the effect of pH, temperature, and light on the stability of betanin. Lastly, the applicability of betanin was taken into account in the eighth section which comprised of dyeing of acrylic fabric, dyeing of wool fabric, and medicinal and food

H NMR, 13C NMR, LC-1

H NMR,

**8.2 Medicinal application**

**8.3 Betanin as food colorant**

**9. Summary**

**186**

Also, it is used in soups as well as bacon and tomato.

in the sixth section which includes UV-Vis, FT-IR, <sup>1</sup>

colorant applications of betanin.

## **Author details**

Deepak Devadiga and T.N. Ahipa\* Centre for Nano and Material Sciences, Jain University, Bangalore, India

\*Address all correspondence to: tn.ahipa@jainuniversity.ac.in

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[40] Sengupta D, Mondal B, Mukherjee K. Visible light absorption and photo-sensitizing properties of spinach leaves and beetroot extracted natural dyes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2015;**148**:85-92

[41] Dong J, Ozaki Y, Nakashima K. Infrared, Raman, and near-infrared spectroscopic evidence for the coexistence of various hydrogen-bond forms in poly(acrylic acid). Macromolecules. 1997;**30**(4):1111-1117

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*DOI: http://dx.doi.org/10.5772/intechopen.88939*

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**53**(23):9268-9273

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[53] Sutariya B, Saraf M. Betanin, isolated from fruits of *Opuntia elatior* Mill attenuates renal fibrosis in diabetic rats through regulating oxidative stress

and TGF-β pathway. Journal of

[54] Amjadi S, Ghorbani M,

by liposomal nanocarriers: Its

156-162

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Ethnopharmacology. 2017;**198**:432-443

Hamishehkar H, Roufegarinejad L. Improvement in the stability of betanin

application in gummy candy as a food model. Food Chemistry. 2018;**256**:

Ameliorating effect of betanin, a natural chromoalkaloid by modulating hepatic carbohydrate metabolic enzyme activities and glycogen content in streptozotocin—Nicotinamide induced experimental rats. Biomedicine & Pharmacotherapy. 2017;**88**:1069-1079

[50] Reddy MK, Alexander-Lindo RL, Nair MG. Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation by

Agricultural and Food Chemistry. 2005;

[51] Krajka-Kuźniak V, Paluszczak J, Szaefer H, Baer-Dubowska W. Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2 mediated expression of detoxifying/ antioxidant enzymes in human liver cell lines. The British Journal of Nutrition.

[42] Stintzing FC, Conrad J, Klaiber I, Beifuss U, Carle R. Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy. Phytochemistry. 2004; **65**(4):415-422

[43] Guesmi A, Ladhari N, Ben Hamadi N, Sakli F. Isolation, identification and dyeing studies of betanin on modified acrylic fabrics. Industrial Crops and Products. 2012; **37**(1):342-346

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[45] Von Elbe JH, Young Maing I, Amundson CH. Color stability os betanin. Journal of Food Science. 1974; **39**(2):334-337

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[47] Khan MI. Plant betalains: Safety, antioxidant activity, clinical efficacy, and bioavailability. Comprehensive Reviews in Food Science and Food Safety. 2016;**15**(2):316-330

[48] Gentile C, Tesoriere L, Allegra M, Livrea MA, D'Alessio P. Antioxidant betalains from cactus pear (*Opuntia ficus-indica*) inhibit endothelial ICAM-1 expression. Annals of the New York Academy of Sciences. 2004;**1028**:481-486

[49] Kapadia GJ, Azuine MA, Rao GS, Arai T, Iida A, Tokuda H. Cytotoxic

*Betanin: A Red-Violet Pigment - Chemistry and Applications DOI: http://dx.doi.org/10.5772/intechopen.88939*

effect of the red beetroot (*Beta vulgaris* L.) extract compared to doxorubicin (Adriamycin) in the human prostate (PC-3) and breast (MCF-7) cancer cell lines. Anti-Cancer Agents in Medicinal Chemistry. 2011;**11**(3):280-284

o'clocks (*Mirabilis jalapa* L.). FEBS Letters. 2004;**568**(1–3):159-162

Totafsynthese von betalainen. Helvetica Chimica Acta. 1975;**58**(6):1805-1808

*Chemistry and Technology of Natural and Synthetic Dyes and Pigments*

[41] Dong J, Ozaki Y, Nakashima K. Infrared, Raman, and near-infrared spectroscopic evidence for the

coexistence of various hydrogen-bond

Macromolecules. 1997;**30**(4):1111-1117

[42] Stintzing FC, Conrad J, Klaiber I,

investigations on betacyanin pigments

spectroscopy. Phytochemistry. 2004;

[44] Susha N, Nandakumar K, Nair SS. Enhanced photoconductivity in CdS/ betanin composite nanostructures. RSC Advances. 2018;**8**(21):11330-11337

[45] Von Elbe JH, Young Maing I, Amundson CH. Color stability os betanin. Journal of Food Science. 1974;

[46] Guesmi A, Ladhari N, Ben Hamadi N, Msaddek M, Sakli F. First application of chlorophyll-a as

Production. 2013;**39**:97-104

Safety. 2016;**15**(2):316-330

biomordant: Sonicator dyeing of wool with betanin dye. Journal of Cleaner

[47] Khan MI. Plant betalains: Safety, antioxidant activity, clinical efficacy, and bioavailability. Comprehensive Reviews in Food Science and Food

[48] Gentile C, Tesoriere L, Allegra M, Livrea MA, D'Alessio P. Antioxidant betalains from cactus pear (*Opuntia ficus-indica*) inhibit endothelial ICAM-1 expression. Annals of the New York Academy of Sciences. 2004;**1028**:481-486

[49] Kapadia GJ, Azuine MA, Rao GS, Arai T, Iida A, Tokuda H. Cytotoxic

forms in poly(acrylic acid).

Beifuss U, Carle R. Structural

[43] Guesmi A, Ladhari N, Ben Hamadi N, Sakli F. Isolation, identification and dyeing studies of betanin on modified acrylic fabrics. Industrial Crops and Products. 2012;

by LC NMR and 2D NMR

**65**(4):415-422

**37**(1):342-346

**39**(2):334-337

[35] Dumbrava A, Enache I, Oprea CI, Georgescu A, Girtu MA. Toward a more efficient utilisation of betalains as pigments for dye-sensitized solar cells. Digest Journal of Nanomaterials and Biostructures. 2012;**7**(1):339-351

[36] Aztatzi-Rugerio L, Granados-Balbuena SY, Zainos-Cuapio Y, Ocaranza-Sánchez E, Rojas-López M. Analysis of the degradation of betanin obtained from beetroot using Fourier transform infrared spectroscopy. Journal of Food Science and Technology. 2019;**56**(8):3677-3686

[37] Kumar SNA, Ritesh SK, Sharmila G,

[38] Cai Y, Sun M, Wu H, Huang R, Corke H. Characterization and

[39] Molina GA, Hernández-Martínez AR, Cortez-Valadez M, García-Hernández F, Estevez M. Effects of tetraethyl orthosilicate (teos) on the light and temperature stability of a pigment from *Beta vulgaris* and its potential food industry applications. Molecules. 2014;**19**(11):17985-18002

[40] Sengupta D, Mondal B,

A: Molecular and Biomolecular Spectroscopy. 2015;**148**:85-92

**190**

Mukherjee K. Visible light absorption and photo-sensitizing properties of spinach leaves and beetroot extracted natural dyes. Spectrochimica Acta Part

quantification of betacyanin pigments from diverse Amaranthus species. Journal of Agricultural and Food Chemistry. 1998;**46**(6):2063-2070

Muthukumaran C. Extraction optimization and characterization of water soluble red purple pigment from floral bracts of Bougainvillea glabra. Arabian Journal of Chemistry. 2017;**10**:

S2145-S2150

[34] Hermama K, Dreiding AS.

[50] Reddy MK, Alexander-Lindo RL, Nair MG. Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation by natural food colors. Journal of Agricultural and Food Chemistry. 2005; **53**(23):9268-9273

[51] Krajka-Kuźniak V, Paluszczak J, Szaefer H, Baer-Dubowska W. Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2 mediated expression of detoxifying/ antioxidant enzymes in human liver cell lines. The British Journal of Nutrition. 2013;**110**(12):2138-2149

[52] Dhananjayan I, Kathiroli S, Subramani S, Veerasamy V. Ameliorating effect of betanin, a natural chromoalkaloid by modulating hepatic carbohydrate metabolic enzyme activities and glycogen content in streptozotocin—Nicotinamide induced experimental rats. Biomedicine & Pharmacotherapy. 2017;**88**:1069-1079

[53] Sutariya B, Saraf M. Betanin, isolated from fruits of *Opuntia elatior* Mill attenuates renal fibrosis in diabetic rats through regulating oxidative stress and TGF-β pathway. Journal of Ethnopharmacology. 2017;**198**:432-443

[54] Amjadi S, Ghorbani M, Hamishehkar H, Roufegarinejad L. Improvement in the stability of betanin by liposomal nanocarriers: Its application in gummy candy as a food model. Food Chemistry. 2018;**256**: 156-162

**193**

Section 5

Printing with Natural

Dyes and Pigments

Section 5
