**Health Benefits**

[49] Lambert P, Dicenta F, Rubio M, Audergon JM. QTL analysis of resistance to Sharka disease in the apricot (*Prunus armeniaca* L.) 'Polonais' x 'stark early Orange' F1 progeny.

[50] Vilanova S, Romero C, Abbott AG, Llacer G, Badenes ML. An apricot (*Prunus armeniaca* L.) F2 progeny linkage map based on SSR and AFLP markers mapping plum pox virus resistance and self-incompatibility traits. Theoretical and Applied Genetics.

[51] Sicard O, Marandel G, Soriano JM, Lalli DA, Lambert P, Salava J, Badenes ML, Abbott A, Decroocq V. Flanking the major plum pox virus resistance locus in apricot with codominant markers (SSRs) derived from candidate resistance genes. Tree Genetics &

[52] DA L, Abbott AG, Zhebentyayeva TN, Badenes ML, Damsteegt V, PoláK J, Krška B, Salava J. A genetic linkage map for an apricot (*Prunus armeniaca* L.) BC1 population

[53] Krška B, Pavelková P, Salava J. Preliminary results on inheritance of resistance to plum pox virus in apricot obtained within gene pyramiding. In: Çağlayan K, editor. Proceedings of the III International Symposium on Plum Pox Virus; Acta Horticulturae.

mapping plum pox virus resistance. Tree Genetics & Genomes. 2008;**4**:481-493

ISHS 2017. 1163. pp. 13-17. DOI: 10.17660/ActaHortic.2017.1163.3

Tree Genetics & Genomes. 2007;**3**:299-309

2003;**107**:239-247

Genomes. 2008;**4**:359-365

82 Breeding and Health Benefits of Fruit and Nut Crops

**Chapter 5**

Provisional chapter

*Myrciaria dubia* **"Camu Camu" Fruit: Health-Promoting**

DOI: 10.5772/intechopen.73213

"Camu Camu" Fruit: Health-Promoting

**Phytochemicals and Functional Genomic Characteristics**

Camu camu is a typical Amazon native fruit shrub that possesses a diploid genome, moderate genetic diversity, and population structure. The fruits accumulate several essential nutrients and synthesize L-ascorbic acid (vitamin C) in great quantities and an array of diverse secondary metabolites with corroborated in vitro and in vivo health-promoting activities. These beneficial effects include antioxidative and antiinflammatory activities, antiobesity, hypolipidemic, antihypertensive and antidiabetic effects, DNA damage and cancer protection effects, and other bioactivities. Many health-promoting phytochemicals are biosynthesized in several metabolic pathways of camu camu. Their reconstruction from the fruit transcriptome database was accomplished by our research group. These include basic metabolic pathways such as glycolysis and pentose phosphate pathway, vitamin C biosynthesis pathways, and pathways involved in secondary metabolites production. Due to their agronomic potential and fruits growing demand, recently, based on an ideotype, programs were initiated for their domestication and genetic improvement, but so far with very negligible achievements. Consequently, we propose new strategies to accelerate the processes of domestication and genetic improvement based on state of the

art technologies for multiomic data analysis and innovative molecular tools.

Keywords: genetic diversity, health-promoting phytochemicals, phenolic compounds,

Myrciaria dubia Kunth (McVaugh) "camu camu" is a typical native Amazonian fruit shrub that thrives in areas exposed to periodical flooding on the banks of rivers, streams, lakes, and swamps

> © 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 eproduction in any medium, provided the original work is properly cited.

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

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

Phytochemicals and Functional Genomic Characteristics

Juan C. Castro, J. Dylan Maddox, Marianela Cobos and Sixto A. Imán

Juan C. Castro, J. Dylan Maddox, Marianela Cobos and Sixto A. Imán

dubia

Myrciaria

http://dx.doi.org/10.5772/intechopen.73213

transcriptome, vitamin C

1. Introduction

Abstract

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### *Myrciaria dubia* **"Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics** Myrciaria dubia "Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics

DOI: 10.5772/intechopen.73213

Juan C. Castro, J. Dylan Maddox, Marianela Cobos and Sixto A. Imán Juan C. Castro, J. Dylan Maddox, Marianela Cobos and Sixto A. Imán

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.73213

#### Abstract

Camu camu is a typical Amazon native fruit shrub that possesses a diploid genome, moderate genetic diversity, and population structure. The fruits accumulate several essential nutrients and synthesize L-ascorbic acid (vitamin C) in great quantities and an array of diverse secondary metabolites with corroborated in vitro and in vivo health-promoting activities. These beneficial effects include antioxidative and antiinflammatory activities, antiobesity, hypolipidemic, antihypertensive and antidiabetic effects, DNA damage and cancer protection effects, and other bioactivities. Many health-promoting phytochemicals are biosynthesized in several metabolic pathways of camu camu. Their reconstruction from the fruit transcriptome database was accomplished by our research group. These include basic metabolic pathways such as glycolysis and pentose phosphate pathway, vitamin C biosynthesis pathways, and pathways involved in secondary metabolites production. Due to their agronomic potential and fruits growing demand, recently, based on an ideotype, programs were initiated for their domestication and genetic improvement, but so far with very negligible achievements. Consequently, we propose new strategies to accelerate the processes of domestication and genetic improvement based on state of the art technologies for multiomic data analysis and innovative molecular tools.

Keywords: genetic diversity, health-promoting phytochemicals, phenolic compounds, transcriptome, vitamin C

#### 1. Introduction

Myrciaria dubia Kunth (McVaugh) "camu camu" is a typical native Amazonian fruit shrub that thrives in areas exposed to periodical flooding on the banks of rivers, streams, lakes, and swamps

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

of several Amazonian countries [1, 2]. This plant species possesses a diploid genome, and their genome size (230 Mb) is in the range of other Myrtaceae species [3–6]. Populations exhibit moderate genetic diversity and genetic structuring [7–11]. Camu camu produces several essential nutrients such as amino acids, polyunsaturated fatty acids, B-complex vitamins, and high quantities of vitamin C [2, 12–15]. Additionally, the fruits (including peel, pulp, and seeds) and several other tissues/organs (leaves, roots, etc.) accumulate numerous health-promoting phytochemicals with powerful antioxidant, antiinflammatory activities, antiobesity, hypolipidemic, antidiabetic effects, DNA damage and cancer protection effects, hepatoprotective properties, and other beneficial effects [16–24]. These bioactive phytochemicals, in addition to vitamin C, are secondary metabolites that primarily include various phenolic compounds, carotenoids, terpenoids, and several other bioactive metabolites. These associated beneficial effects of phytochemicals were corroborated by numerous in vitro and in vivo studies with several animal models (i.e., flies, mice, rats, etc.) and human volunteers [16, 17, 22, 24–28]. In the healthpromoting phytochemicals section of the book chapter, we will illustrate the chemical structures of several of these phytochemicals that were isolated from various tissues of camu camu by bioassay-guided approaches. These secondary metabolites were biosynthesized in several metabolic pathways of camu camu. In the functional genomic characteristics section, we have presented the reconstruction of some metabolic pathways from the fruit transcriptome database that was accomplished by our research group. These include, for example, basic metabolic pathways (i.e., glycolysis, pentose phosphate pathway), vitamin C biosynthesis pathways, shikimate pathway, and pathways directly involved in secondary metabolites production (i.e., anthocyanins, carotenoids, flavonoids, phenylpropanoids, and terpenoids biosynthesis pathways). Finally, we have included a section about domestication strategy and genetic improvement efforts, where we examine the strategies implemented by Peruvian Institutions to achieve these goals. We have also proposed new strategies to significantly accelerate the domestication and genetic improvement of this species based on the state of the art technologies for multiomic data analysis and innovative molecular tools.

#### 2. General description

#### 2.1. Geographical distribution

Camu camu is a typical native shrub from the tropical rainforest of the Amazon. Wild populations of this species grow in dense areas exposed to periodical flooding (complete submergence for 4–5 months) on the banks of rivers, streams, lakes, and swamps of Guyana, Venezuela (Casiquiare Oreda, Pargueni, Caura, and Orinoco), Colombia (Putumayo and Inirida), Ecuador, Brazil (Trombetas, Cachorro, Mapuera, Maçangana, Urupa, Javari, Solimões, Madeira, and Negro), Peru (Amazonas, Curaray, Itaya, Nanay, Napo, Putumato, Ucayali, Marañon, and Tigre), and Bolivia (Figure 1) [1, 2]. In Peru, wild populations only exist in the Loreto Region, consisting of approximately 1345 ha [29], whereas artificial plantations have been established in the Regions Loreto (6475 ha), San Martin (110 ha), and Ucayali that consist of approximately 5930 ha [29–32].

Figure 1. Geographical distribution of camu camu in South American and the Peruvian Loreto region.

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*Myrciaria dubia* "Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics http://dx.doi.org/10.5772/intechopen.73213 87

of several Amazonian countries [1, 2]. This plant species possesses a diploid genome, and their genome size (230 Mb) is in the range of other Myrtaceae species [3–6]. Populations exhibit moderate genetic diversity and genetic structuring [7–11]. Camu camu produces several essential nutrients such as amino acids, polyunsaturated fatty acids, B-complex vitamins, and high quantities of vitamin C [2, 12–15]. Additionally, the fruits (including peel, pulp, and seeds) and several other tissues/organs (leaves, roots, etc.) accumulate numerous health-promoting phytochemicals with powerful antioxidant, antiinflammatory activities, antiobesity, hypolipidemic, antidiabetic effects, DNA damage and cancer protection effects, hepatoprotective properties, and other beneficial effects [16–24]. These bioactive phytochemicals, in addition to vitamin C, are secondary metabolites that primarily include various phenolic compounds, carotenoids, terpenoids, and several other bioactive metabolites. These associated beneficial effects of phytochemicals were corroborated by numerous in vitro and in vivo studies with several animal models (i.e., flies, mice, rats, etc.) and human volunteers [16, 17, 22, 24–28]. In the healthpromoting phytochemicals section of the book chapter, we will illustrate the chemical structures of several of these phytochemicals that were isolated from various tissues of camu camu by bioassay-guided approaches. These secondary metabolites were biosynthesized in several metabolic pathways of camu camu. In the functional genomic characteristics section, we have presented the reconstruction of some metabolic pathways from the fruit transcriptome database that was accomplished by our research group. These include, for example, basic metabolic pathways (i.e., glycolysis, pentose phosphate pathway), vitamin C biosynthesis pathways, shikimate pathway, and pathways directly involved in secondary metabolites production (i.e., anthocyanins, carotenoids, flavonoids, phenylpropanoids, and terpenoids biosynthesis pathways). Finally, we have included a section about domestication strategy and genetic improvement efforts, where we examine the strategies implemented by Peruvian Institutions to achieve these goals. We have also proposed new strategies to significantly accelerate the domestication and genetic improvement of this species based on the state of the art technologies for multiomic

Camu camu is a typical native shrub from the tropical rainforest of the Amazon. Wild populations of this species grow in dense areas exposed to periodical flooding (complete submergence for 4–5 months) on the banks of rivers, streams, lakes, and swamps of Guyana, Venezuela (Casiquiare Oreda, Pargueni, Caura, and Orinoco), Colombia (Putumayo and Inirida), Ecuador, Brazil (Trombetas, Cachorro, Mapuera, Maçangana, Urupa, Javari, Solimões, Madeira, and Negro), Peru (Amazonas, Curaray, Itaya, Nanay, Napo, Putumato, Ucayali, Marañon, and Tigre), and Bolivia (Figure 1) [1, 2]. In Peru, wild populations only exist in the Loreto Region, consisting of approximately 1345 ha [29], whereas artificial plantations have been established in the Regions Loreto (6475 ha), San Martin (110 ha), and Ucayali that consist of approximately 5930 ha [29–32].

data analysis and innovative molecular tools.

86 Breeding and Health Benefits of Fruit and Nut Crops

2. General description

2.1. Geographical distribution

Figure 1. Geographical distribution of camu camu in South American and the Peruvian Loreto region.

#### 2.2. Botanical characteristics

Typically, the camu camu shrub achieves a height of 4–8 m, branching from the base to form several secondary stems, which in turn branch out as an open vessel. The trunk and branches are glabrous, cylindrical, and smooth, and the bark is light or reddish brown, which peels off naturally in periods of drought [14, 33]. The deep-rooted shrub contains numerous absorbing roots. The leaves are opposed, single, petiolar, elliptic-lanceolate (ca. 3–12 1.5–4.5 cm), with acuminated apex and oval base, with primary and secondary veins (18–20 pairs). Petioles that are cylindrical have a length of 3–9 1–2 mm [34]. The inflorescences are axillary with 1–12 (generally four) subsessile and hermaphrodite flowers arranged in two pairs on the axis. The rounded ciliated bracts and bracteoles are persistent. The calyx is approximately 2 mm long and 2 mm wide and includes four sepals with broad apex and the hypanthium is prolonged and circumscissile at the summit of the ovary and falls with the calyx as a unit after anthesis [35]. The corolla has four white ovate petals which are 3–4 mm long with a ciliated margin. The ovary is inferior with a simple style that is 10–11 mm long, and the androecium has 125 stamens of 6– 10 mm in length and anthers of 0.5–0.7 mm length. Although camu camu flowers are hermaphrodite, inbreeding is largely prevented by the lack of synchrony between the development of the gynoecium and androecium, leading to facultative allogamy [14, 33, 36]. The fruits are globular and measure 1.0–5.0 cm in diameter, and their weight averages 11.7 1.4 g [34]. Based on the fresh weight, the fruits are comprised, on an average, of 65.2% pulp, 19.5% seeds, and 15.3% peel [34, 37]. The shiny peel can be pink to deep red or even black when completely ripe, with slightly pinkish pulp [2, 14, 33]. The seeds are kidney-shaped to ellipsoid, flattened bilaterally and are exalbuminous. The fruit contains one to four seeds with an average length of 13.5 1.6 and width of 4.8 0.6 mm. The average fresh seed weight is 440 170 mg. The elongated seed coats are brown and thin and are covered with spiny-celled villi (Figure 2) [38, 39].

#### 2.3. Nutritional composition

Pioneering work on the high L-ascorbic acid (vitamin C) content of camu camu fruits was published in 1964 [15]. In this report, the authors indicate that camu camu fruits are among the highest natural sources of vitamin C. Approximately 30 years later, these observations were corroborated by several investigations on the chemical and nutritional composition of camu camu [2, 12, 13, 34, 37, 40–44]. These findings from approximately 60 years of camu camu research are consolidated in Table 1. These fruits are composed of nutrients such as protein, carbohydrate, lipids, ash, and crude fiber. Additionally, they have essential amino acids (valine, leucine, phenylalanine, etc.), essential fatty acids of the families omega 3 and 6, vitamin C, and vitamins of the B-complex and several essential minerals for human nutrition, such as potassium, phosphorous, sulfate, calcium, magnesium, cobalt, iron, and several others.

grandis, Gomidesia affinis, etc.) [5]. Using flow cytometry, our research group recently determined that the genome size of camu camu is 230 Mb (unpublished data), which is similar to Myrciaria glazioviana (234 Mb) [6] but is on the lower end of the range (234–1110 Mb) reported for Myrtaceae

Figure 2. Botanical characteristics of camu camu and harvest strategies. Wild populations in flooding soils (A) and nonflooding soils (B), culture population in nonflooding soils (C), blooming flowers (D), unripe, semiripe, and ripe fruits (E), variations in seeds size (F), typical plant height and architecture (G), manual harvest during flooding period using

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89

Pioneering research analyzing the genetic diversity of camu camu was carried out with biochemical markers (esterases) by Brazilian researchers [7]. At that time, the presence of genetic structure was demonstrated among populations (two genetic groups) with an average heterozygosity of 0.08–0.14. Subsequent studies have analyzed the genetic diversity in germplasm collections and cultured populations using DNA markers, such as rapid amplification of polymorphic DNA (RAPD) [45], inter simple sequence repeat (ISSR) [8], expressed sequences tag-simple sequence repeats (EST-SSR) [9, 46, 47], and SSR [10, 11], also known as microsatellite markers. Overall, these investigations found that the average expected heterozygosity (He = 0.67 0.19, range of 0.45–0.88) was greater than the average observed heterozygosity

species [5].

2.5. Genetic diversity

canoes (H), and manual harvest in non-flooding soils.

#### 2.4. Chromosome number and genome size

Some standardized karyotype analyses conducted on meristematic cells from root apices have demonstrated that camu camu is a diploid plant with 2n = 22 chromosomes [3, 4], which is consistent with several other Myrtaceae species (i.e., Acca sellowiana, Callistemon citrinus, Eucalyptus

*Myrciaria dubia* "Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics http://dx.doi.org/10.5772/intechopen.73213 89

Figure 2. Botanical characteristics of camu camu and harvest strategies. Wild populations in flooding soils (A) and nonflooding soils (B), culture population in nonflooding soils (C), blooming flowers (D), unripe, semiripe, and ripe fruits (E), variations in seeds size (F), typical plant height and architecture (G), manual harvest during flooding period using canoes (H), and manual harvest in non-flooding soils.

grandis, Gomidesia affinis, etc.) [5]. Using flow cytometry, our research group recently determined that the genome size of camu camu is 230 Mb (unpublished data), which is similar to Myrciaria glazioviana (234 Mb) [6] but is on the lower end of the range (234–1110 Mb) reported for Myrtaceae species [5].

#### 2.5. Genetic diversity

2.2. Botanical characteristics

88 Breeding and Health Benefits of Fruit and Nut Crops

2.3. Nutritional composition

Typically, the camu camu shrub achieves a height of 4–8 m, branching from the base to form several secondary stems, which in turn branch out as an open vessel. The trunk and branches are glabrous, cylindrical, and smooth, and the bark is light or reddish brown, which peels off naturally in periods of drought [14, 33]. The deep-rooted shrub contains numerous absorbing roots. The leaves are opposed, single, petiolar, elliptic-lanceolate (ca. 3–12 1.5–4.5 cm), with acuminated apex and oval base, with primary and secondary veins (18–20 pairs). Petioles that are cylindrical have a length of 3–9 1–2 mm [34]. The inflorescences are axillary with 1–12 (generally four) subsessile and hermaphrodite flowers arranged in two pairs on the axis. The rounded ciliated bracts and bracteoles are persistent. The calyx is approximately 2 mm long and 2 mm wide and includes four sepals with broad apex and the hypanthium is prolonged and circumscissile at the summit of the ovary and falls with the calyx as a unit after anthesis [35]. The corolla has four white ovate petals which are 3–4 mm long with a ciliated margin. The ovary is inferior with a simple style that is 10–11 mm long, and the androecium has 125 stamens of 6– 10 mm in length and anthers of 0.5–0.7 mm length. Although camu camu flowers are hermaphrodite, inbreeding is largely prevented by the lack of synchrony between the development of the gynoecium and androecium, leading to facultative allogamy [14, 33, 36]. The fruits are globular and measure 1.0–5.0 cm in diameter, and their weight averages 11.7 1.4 g [34]. Based on the fresh weight, the fruits are comprised, on an average, of 65.2% pulp, 19.5% seeds, and 15.3% peel [34, 37]. The shiny peel can be pink to deep red or even black when completely ripe, with slightly pinkish pulp [2, 14, 33]. The seeds are kidney-shaped to ellipsoid, flattened bilaterally and are exalbuminous. The fruit contains one to four seeds with an average length of 13.5 1.6 and width of 4.8 0.6 mm. The average fresh seed weight is 440 170 mg. The elongated seed coats

are brown and thin and are covered with spiny-celled villi (Figure 2) [38, 39].

phosphorous, sulfate, calcium, magnesium, cobalt, iron, and several others.

2.4. Chromosome number and genome size

Pioneering work on the high L-ascorbic acid (vitamin C) content of camu camu fruits was published in 1964 [15]. In this report, the authors indicate that camu camu fruits are among the highest natural sources of vitamin C. Approximately 30 years later, these observations were corroborated by several investigations on the chemical and nutritional composition of camu camu [2, 12, 13, 34, 37, 40–44]. These findings from approximately 60 years of camu camu research are consolidated in Table 1. These fruits are composed of nutrients such as protein, carbohydrate, lipids, ash, and crude fiber. Additionally, they have essential amino acids (valine, leucine, phenylalanine, etc.), essential fatty acids of the families omega 3 and 6, vitamin C, and vitamins of the B-complex and several essential minerals for human nutrition, such as potassium,

Some standardized karyotype analyses conducted on meristematic cells from root apices have demonstrated that camu camu is a diploid plant with 2n = 22 chromosomes [3, 4], which is consistent with several other Myrtaceae species (i.e., Acca sellowiana, Callistemon citrinus, Eucalyptus Pioneering research analyzing the genetic diversity of camu camu was carried out with biochemical markers (esterases) by Brazilian researchers [7]. At that time, the presence of genetic structure was demonstrated among populations (two genetic groups) with an average heterozygosity of 0.08–0.14. Subsequent studies have analyzed the genetic diversity in germplasm collections and cultured populations using DNA markers, such as rapid amplification of polymorphic DNA (RAPD) [45], inter simple sequence repeat (ISSR) [8], expressed sequences tag-simple sequence repeats (EST-SSR) [9, 46, 47], and SSR [10, 11], also known as microsatellite markers. Overall, these investigations found that the average expected heterozygosity (He = 0.67 0.19, range of 0.45–0.88) was greater than the average observed heterozygosity


(Ho = 0.41 0.06, range of 0.33–0.49). Also, the populations exhibited high inbreeding coefficients (f = 0.31 0.13, range of 0.20–0.49) and high genetic differentiation values (FST = 0.26 0.08, range of 0.21–0.32). Likewise, the average intrapopulation genetic diversity (average 74.89%, range of 65–79%) is 3 times greater than average interpopulation genetic diversity (average 25.10%, range of 20–35%). Also, these molecular markers studies demonstrated the presence of genetic structure among populations (from 2 to 3 genetic groups). However, two of these reports have shown that genetic and geographic distances are uncorrelated (r = 0.31, range of 0.23–0.39). These peculiar genetic population characteristics of camu camu can be partially explained as the result of their undomesticated condition and isolation of the populations by natural barriers, which limit gene flow and favor inbreeding. In conclusion, the genetic diversity characterization of camu camu in wild and artificial populations, and germplasm banks are still too fragmented and deficient to make any strong conclusions. Consequently, an in-depth knowledge of the genetic diversity of this species will be essential to implement programs for genome-wide genetic marker discovery and genotyping using next-generation sequencing technologies, which could then be used to quantify, with more precision and accuracy, the genetic diversity of camu camu across the entire Amazon region. Following this line of thought, our research group explored the assembled transcriptome of this species for molecular marker discovery. We identified more than 3200 SSR motifs that would be appropriate for developing a comprehensive set of genic-SSR markers. Also, the transcriptome contained a large number (>23,000) of high-quality singlenucleotide polymorphisms (SNPs) and marks the highest number of SNP markers discovered to date for camu camu using transcriptome sequencing [48]. Both types of potential molecular

Component per 100 g Contents

Al 0.255 0.064 Zn 0.230 0.138 Cu 0.117 0.072 B 0.050 0.000 Br 0.021 0.005 Cr 0.015 0.004 Mo 0.004 0.002 Se (μg) 0.429 0.089

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An ethnopharmacological survey of medicinal plants in the northeastern Amazon region of Peru showed that several botanical parts of camu camu such as immature and mature fruits, stems, leaves, roots, seeds, and barks are used to prepare remedies in folk medicine to treat numerous diseases such as arthritis, diabetes, hypercholesterolemia, bronchitis, inflammation,

markers, however, will require validation.

3. Health-promoting phytochemicals

Table 1. Nutritional composition of camu camu fruit pulp.

*Myrciaria dubia* "Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics http://dx.doi.org/10.5772/intechopen.73213 91


Table 1. Nutritional composition of camu camu fruit pulp.

Component per 100 g Contents

Energy (kcal) 19.48 3.68 Water 93.83 0.51 Protein 0.51 0.07 Carbohydrate 4.84 0.80 Lipids 0.17 0.10 Ash 0.22 0.03 Crude fiber 0.56 0.40 Total soluble solids (Brix) 6.18 0.99 pH 2.84 0.31

Valine 242.00 104.65 Leucine 210.50 111.02 Phenylalanine 32.50 14.85 Threonine 32.00 5.66

C18:3ω3 (α-Linolenic) 16.00 0.70 C18:2ω6 (Linoleic) 9.70 0.40 C18:3ω6 (γ-Linolenic) 9.30 0.20 C20:5ω3 (EPA) 7.00 0.10

Vitamin C 2210.00 650.00 Niacin 0.48 0.28 Riboflavin 0.03 0.02 Thiamine 0.01 0.00

K 87.020 29.382 PO4 18.183 8.122 SO4 14.750 2.192 Ca 14.510 9.346 Cl 9.100 3.536 Mg 7.393 4.323 Co 1.173 0.807 Na 0.934 1.546 Mn 0.820 1.118 Fe 0.424 0.152

Bromatological analysis

90 Breeding and Health Benefits of Fruit and Nut Crops

Essential amino acids (mg/100 g)

Essential fatty acids (% of total lipids)

Vitamins (mg/100 g)

Minerals (mg/100 g)

(Ho = 0.41 0.06, range of 0.33–0.49). Also, the populations exhibited high inbreeding coefficients (f = 0.31 0.13, range of 0.20–0.49) and high genetic differentiation values (FST = 0.26 0.08, range of 0.21–0.32). Likewise, the average intrapopulation genetic diversity (average 74.89%, range of 65–79%) is 3 times greater than average interpopulation genetic diversity (average 25.10%, range of 20–35%). Also, these molecular markers studies demonstrated the presence of genetic structure among populations (from 2 to 3 genetic groups). However, two of these reports have shown that genetic and geographic distances are uncorrelated (r = 0.31, range of 0.23–0.39). These peculiar genetic population characteristics of camu camu can be partially explained as the result of their undomesticated condition and isolation of the populations by natural barriers, which limit gene flow and favor inbreeding. In conclusion, the genetic diversity characterization of camu camu in wild and artificial populations, and germplasm banks are still too fragmented and deficient to make any strong conclusions. Consequently, an in-depth knowledge of the genetic diversity of this species will be essential to implement programs for genome-wide genetic marker discovery and genotyping using next-generation sequencing technologies, which could then be used to quantify, with more precision and accuracy, the genetic diversity of camu camu across the entire Amazon region. Following this line of thought, our research group explored the assembled transcriptome of this species for molecular marker discovery. We identified more than 3200 SSR motifs that would be appropriate for developing a comprehensive set of genic-SSR markers. Also, the transcriptome contained a large number (>23,000) of high-quality singlenucleotide polymorphisms (SNPs) and marks the highest number of SNP markers discovered to date for camu camu using transcriptome sequencing [48]. Both types of potential molecular markers, however, will require validation.

#### 3. Health-promoting phytochemicals

An ethnopharmacological survey of medicinal plants in the northeastern Amazon region of Peru showed that several botanical parts of camu camu such as immature and mature fruits, stems, leaves, roots, seeds, and barks are used to prepare remedies in folk medicine to treat numerous diseases such as arthritis, diabetes, hypercholesterolemia, bronchitis, inflammation, asthma, atherosclerosis, cataracts, depression, flu, gingivitis, glaucoma, hepatitis, infertility, migraine, osteoporosis, Parkinson's disease, and malaria [49, 50]. Additionally, Steele [51] showed that camu camu is used traditionally for the treatment of malaria by indigenous people of South America. All these traditional uses are in concordance with multiple scientific researches showing that several botanical parts of camu camu are a rich source of various health-promoting phytochemicals with proved health beneficial properties. Among these bioactive phytochemicals, in addition to vitamin C, several secondary metabolites exist such as polyphenols, carotenoids, and other chemicals, which are presented in Figures 3 and 4 and detailed below.

#### 3.1. Antioxidative and antiinflammatory activities

A large amount of scientific information currently exists regarding the antioxidant properties of camu camu fruits that were collected by diverse methods, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH assay), 2,2<sup>0</sup> -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing ability of plasma (FRAP assay), oxygen radical absorbance capacity assay (ORAC assay), total radical-trapping antioxidant parameter (TRAP assay), β-carotene bleaching method, cupric ion reducing antioxidant capacity (CUPRAC assay), total oxidant scavenging capacity (TOSC assay), Trolox equivalent antioxidant capacity (TEAC assay), peroxyl radical scavenging capacity (PSC assay), and pulse voltammetry measurements (voltammetric electronic tongues) [16, 17, 21, 52– 54]. Pioneering work on antioxidant properties of camu camu was realized by Reynertson et al. [25], who obtained a IC50 value of 57.2 μg/mL on the dried, powdered fruit with the DPPH assay. This low value indicates large antiradical activity, and compared with other Myrtaceae fruits, it was considered very active. These properties were attributed to the high content of vitamin C and total phenolic phytochemicals (101 � 0.25 mg gallic acid equivalent/g dry weight). Furthermore, Sotero et al. [55] reported that a methanolic extract of fruit pulp, fruit peel, and seeds have antioxidant activities with IC50 values of 167.7, 146.9, and 399.8 μg/mL, respectively, with the DPPH assay. Also, De Souza Schmidt Gonçalves et al. [56] demonstrated that lyophilized pulp presented the highest antioxidant capacity with the DPPH assay (�1450 μmol trolox equivalent/g dry weight) and ORAC assay (�800 μmol trolox equivalent/g dry weight), which was ≥10 times higher than 21 other native Brazilian fruits analyzed. Positive correlations were high and significant (r = 0.989 [DPPH vs. total phenolics content], and r = 0.795 [ORAC vs. total phenolics content]) between the antioxidant capacity and total phenolics content (�285 mg catechin equivalent/g dry weight). Additionally, in the fresh fruit pulp, Sánchez [57] found antioxidant capacity values of 219.7 μmol trolox equivalent/g fresh weight and 214.1 μmol trolox equivalent/g fresh weight with the DPPH and ABTS assays, respectively. Another study conducted by Villanueva-Tiburcio et al. [52] compared the antioxidant activity in fruit peel of three maturation stages (unripe, semiripe, and ripe). Semiripe fruits showed the highest antiradical power with the DPPH (IC50 = 46.20 μg/mL), ABTS (IC50 = 20.25 μg/mL), and PSC (IC50 = 8.30 μg/mL) assays. Again, the authors corroborated a highly positive correlation of vitamin C and polyphenol content with the ability to inhibit the free radical DPPH (r = 0.999 with both compounds). Several other investigations have corroborated the highly positive correlations between polyphenol content and antioxidant activity with different assays. Likewise, the superior antioxidant capacity of camu camu fruits was established by the comparison

Figure 3. Some phytochemical compounds with antioxidant activities identified in fruits of camu camu.

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asthma, atherosclerosis, cataracts, depression, flu, gingivitis, glaucoma, hepatitis, infertility, migraine, osteoporosis, Parkinson's disease, and malaria [49, 50]. Additionally, Steele [51] showed that camu camu is used traditionally for the treatment of malaria by indigenous people of South America. All these traditional uses are in concordance with multiple scientific researches showing that several botanical parts of camu camu are a rich source of various health-promoting phytochemicals with proved health beneficial properties. Among these bioactive phytochemicals, in addition to vitamin C, several secondary metabolites exist such as polyphenols, carotenoids, and other chemicals, which are presented in Figures 3 and 4 and

A large amount of scientific information currently exists regarding the antioxidant properties of camu camu fruits that were collected by diverse methods, such as 2,2-diphenyl-1-picrylhydrazyl

ability of plasma (FRAP assay), oxygen radical absorbance capacity assay (ORAC assay), total radical-trapping antioxidant parameter (TRAP assay), β-carotene bleaching method, cupric ion reducing antioxidant capacity (CUPRAC assay), total oxidant scavenging capacity (TOSC assay), Trolox equivalent antioxidant capacity (TEAC assay), peroxyl radical scavenging capacity (PSC assay), and pulse voltammetry measurements (voltammetric electronic tongues) [16, 17, 21, 52– 54]. Pioneering work on antioxidant properties of camu camu was realized by Reynertson et al. [25], who obtained a IC50 value of 57.2 μg/mL on the dried, powdered fruit with the DPPH assay. This low value indicates large antiradical activity, and compared with other Myrtaceae fruits, it was considered very active. These properties were attributed to the high content of vitamin C and total phenolic phytochemicals (101 � 0.25 mg gallic acid equivalent/g dry weight). Furthermore, Sotero et al. [55] reported that a methanolic extract of fruit pulp, fruit peel, and seeds have antioxidant activities with IC50 values of 167.7, 146.9, and 399.8 μg/mL, respectively, with the DPPH assay. Also, De Souza Schmidt Gonçalves et al. [56] demonstrated that lyophilized pulp presented the highest antioxidant capacity with the DPPH assay (�1450 μmol trolox equivalent/g dry weight) and ORAC assay (�800 μmol trolox equivalent/g dry weight), which was ≥10 times higher than 21 other native Brazilian fruits analyzed. Positive correlations were high and significant (r = 0.989 [DPPH vs. total phenolics content], and r = 0.795 [ORAC vs. total phenolics content]) between the antioxidant capacity and total phenolics content (�285 mg catechin equivalent/g dry weight). Additionally, in the fresh fruit pulp, Sánchez [57] found antioxidant capacity values of 219.7 μmol trolox equivalent/g fresh weight and 214.1 μmol trolox equivalent/g fresh weight with the DPPH and ABTS assays, respectively. Another study conducted by Villanueva-Tiburcio et al. [52] compared the antioxidant activity in fruit peel of three maturation stages (unripe, semiripe, and ripe). Semiripe fruits showed the highest antiradical power with the DPPH (IC50 = 46.20 μg/mL), ABTS (IC50 = 20.25 μg/mL), and PSC (IC50 = 8.30 μg/mL) assays. Again, the authors corroborated a highly positive correlation of vitamin C and polyphenol content with the ability to inhibit the free radical DPPH (r = 0.999 with both compounds). Several other investigations have corroborated the highly positive correlations between polyphenol content and antioxidant activity with different assays. Likewise, the superior antioxidant capacity of camu camu fruits was established by the comparison


detailed below.

(DPPH assay), 2,2<sup>0</sup>

3.1. Antioxidative and antiinflammatory activities

92 Breeding and Health Benefits of Fruit and Nut Crops

Figure 3. Some phytochemical compounds with antioxidant activities identified in fruits of camu camu.

of other plant species [16, 17, 19, 58–60]. An in-depth analysis of phenolics compounds was realized by Fracassetti et al. [17]. These researchers identified 53 different phenolics (flavonols [6], anthocyanins [1], ellagic acid and derivatives [10], ellagitannins [10], gallic acid and derivatives [2], and proanthocyanidins [24]) and found differential quantities of these compounds in fruit pulp, peel, seeds, and two powder products from camu camu fruits. Interestingly, the flour produced from the remaining peel, seeds, and adhered pulp after pulp extraction showed a superior vitamin C and phenolics content than the pulp powder (2.6 and 13.9 times, respectively). In addition, the flour displayed greater antioxidant capacity than the pulp power (from

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In vivo studies using Rattus norvegicus (Wistar strain) as an experimental model that were treated orally with fruit pulp and tropical juice containing 5% of camu camu fruit pulp demonstrated a significant increase in plasma antioxidant capacity, liver glutathione peroxidase, superoxide dismutase, and catalase activities and thiobarbituric acid-reactive substances were reduced when compared with control [61, 62]. Finally, Inoue et al. [22] analyzed the antioxidant and antiinflamatory activities of camu camu juice in 10 healthy habitual smokers. The participants ingested 70 mL of juice (100% fruit pulp) daily for 7 days. After the experimental period, the oxidative stress markers such as levels of urinary 8-hydroxy-deoxyguanosine and total reactive oxygen species decreased significantly (p < 0.01) in the camu camu group. Similar trends were evident with inflammatory markers such as serum levels of high-sensitivity C reactive protein, interleukin 6, and interleukin 8, while the group that ingested vitamin C tablets remained unchanged. The researchers concluded that the fruit juice of camu camu may have powerful antioxidant and antiinflammatory properties compared to vitamin C tablets, due to the existence of unknown antioxidant substances modulating in vivo vitamin C in camu camu. These health-promoting activities can be partially explained based on current evidence that the ingestion of the fruit pulp and other derived products significantly increase the postprandial antioxi-

dant capacity of the plasma [63] due to the high vitamin C and polyphenolics content.

3.2. Antiobesity, hypolipidemic, and antihypertensive activities

Additionally, in previous animal experiments using Mus musculus as carrageenan-induced paw edema model, a Japanese research group demonstrated that the methanolic extract of seeds significantly reduced edema formation with regard to size and volume in a dosedependent manner. These antiinflammatory effects were associated with inhibition of the localized nitric oxide production by macrophages. Further, by bioassay-guided fractionation, the researchers identified the active compound to be 3β-hydroxy-lup-20(29)-en-28-oic acid

Several studies in animal models and humans have corroborated the beneficial effects of camu camu fruits on the improvement of biochemical lipid profiles. For example, in animal experiments conducted by Schwertz et al. [24], Wistar rats (R. norvegicus) were induced to a dyslipidemic condition by a high-fat diet and then were subjected to various treatments with camu camu fruit juice (0.4–10 mL/kg) for 2 weeks. All dosages showed an improvement in the biochemical lipid profile in a dose-dependent manner, which was evident by a significant reduction in triacylglycerols, total cholesterol, and hepatic cholesterol. Further, fecal cholesterol

2.0 to 4.5 fold).

(betulinic acid) [23].

Figure 4. Phytochemical compounds with corroborated health-promoting phytochemicals isolated by bioassay-guided approaches from camu camu tissues.

of other plant species [16, 17, 19, 58–60]. An in-depth analysis of phenolics compounds was realized by Fracassetti et al. [17]. These researchers identified 53 different phenolics (flavonols [6], anthocyanins [1], ellagic acid and derivatives [10], ellagitannins [10], gallic acid and derivatives [2], and proanthocyanidins [24]) and found differential quantities of these compounds in fruit pulp, peel, seeds, and two powder products from camu camu fruits. Interestingly, the flour produced from the remaining peel, seeds, and adhered pulp after pulp extraction showed a superior vitamin C and phenolics content than the pulp powder (2.6 and 13.9 times, respectively). In addition, the flour displayed greater antioxidant capacity than the pulp power (from 2.0 to 4.5 fold).

In vivo studies using Rattus norvegicus (Wistar strain) as an experimental model that were treated orally with fruit pulp and tropical juice containing 5% of camu camu fruit pulp demonstrated a significant increase in plasma antioxidant capacity, liver glutathione peroxidase, superoxide dismutase, and catalase activities and thiobarbituric acid-reactive substances were reduced when compared with control [61, 62]. Finally, Inoue et al. [22] analyzed the antioxidant and antiinflamatory activities of camu camu juice in 10 healthy habitual smokers. The participants ingested 70 mL of juice (100% fruit pulp) daily for 7 days. After the experimental period, the oxidative stress markers such as levels of urinary 8-hydroxy-deoxyguanosine and total reactive oxygen species decreased significantly (p < 0.01) in the camu camu group. Similar trends were evident with inflammatory markers such as serum levels of high-sensitivity C reactive protein, interleukin 6, and interleukin 8, while the group that ingested vitamin C tablets remained unchanged. The researchers concluded that the fruit juice of camu camu may have powerful antioxidant and antiinflammatory properties compared to vitamin C tablets, due to the existence of unknown antioxidant substances modulating in vivo vitamin C in camu camu. These health-promoting activities can be partially explained based on current evidence that the ingestion of the fruit pulp and other derived products significantly increase the postprandial antioxidant capacity of the plasma [63] due to the high vitamin C and polyphenolics content.

Additionally, in previous animal experiments using Mus musculus as carrageenan-induced paw edema model, a Japanese research group demonstrated that the methanolic extract of seeds significantly reduced edema formation with regard to size and volume in a dosedependent manner. These antiinflammatory effects were associated with inhibition of the localized nitric oxide production by macrophages. Further, by bioassay-guided fractionation, the researchers identified the active compound to be 3β-hydroxy-lup-20(29)-en-28-oic acid (betulinic acid) [23].

#### 3.2. Antiobesity, hypolipidemic, and antihypertensive activities

Figure 4. Phytochemical compounds with corroborated health-promoting phytochemicals isolated by bioassay-guided

approaches from camu camu tissues.

94 Breeding and Health Benefits of Fruit and Nut Crops

Several studies in animal models and humans have corroborated the beneficial effects of camu camu fruits on the improvement of biochemical lipid profiles. For example, in animal experiments conducted by Schwertz et al. [24], Wistar rats (R. norvegicus) were induced to a dyslipidemic condition by a high-fat diet and then were subjected to various treatments with camu camu fruit juice (0.4–10 mL/kg) for 2 weeks. All dosages showed an improvement in the biochemical lipid profile in a dose-dependent manner, which was evident by a significant reduction in triacylglycerols, total cholesterol, and hepatic cholesterol. Further, fecal cholesterol excretion was increased. In addition, assays performed by Nascimento et al. [44] in a rat model of diet-induced obesity that ingested daily 25 mL of camu camu fruit pulp by 12 weeks resulted in a significant weights reduction of the fat in white adipose tissues. Triacylglycerols (↓40.6%), total cholesterol (↓39.6%), LDL cholesterol (↓2.14%), and VLDL cholesterol (↓36.4%) levels were also decreased, and HDL cholesterol (↑12.3%) increased in experimental groups. Similar experiments conducted by De Souza Schmidt Gonçalves et al. [61] in a type 1 diabetic rat model (streptozotocin induced) receiving oral administration of 1 or 3 g/kg by body weight of aqueous fruit pulp extract by 30 days, significantly reduced triacylglycerols and total cholesterol levels, and an increase in HDL cholesterol. Additionally, some controlled clinical trials with healthy young adults (20–35 years old) who received oral administration of pulp nectar [64] or encapsulated freeze-dried pulp for approximately 2 weeks [65, 66] displayed a significant improvement in their biochemical lipid profiles, due to hypolipidemic effects. The improvement in the biochemical profile of obesity is associated with an increase in lipid elimination in feces (50%) and by the liver (140%) due to the existence of dietary fiber in the fruit pulp [2, 41]. Dietary fibers have shown multiple beneficial health properties by their role in energy intake regulation and obesity development, which are related to its peculiar physicochemical characteristics. Some mechanisms suggested how dietary fiber aids in weight management include promoting satiation, extended signals of satiety, decreasing transport of macronutrients, and altering secretion of gastrointestinal hormones [67–71].

(31%) after a carbohydrate-rich meal ingestion. The hypoglycemic activity of these preparations can be attributed to polyphenols of the camu camu fruits. Polyphenols have two corroborated hypoglycemic mechanisms. The first is related to inhibitory action of the carbohydrate digestive enzymes alpha-amylase and alpha-glucosidase [26, 44, 56, 63, 72] because these inhibitory activities have high-positive correlations (Pearson's correlation coefficient ≥ 0.50) with the concentration of different phenolic compounds (casuarictin, ellagic acid, quercetin, syringic acid, myricetin, etc.) [26, 56]. Second, intestinal monosaccharides transporters such as sodiumdependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) were inhibited,

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Additionally, Ueda et al. [80] isolated three aldose reductase (AR) inhibitors from an 80% methanolic leaf extract: ellagic acid, 4-O-methylellagic acid, and 4-(α-rhamnopyranosyl)ellagic acid. The last phenolic compound showed the strongest uncompetitive inhibition against human recombinant AR and rat lens AR. Also, the inhibitory activity of 4-(α-rhamnopyranosyl)ellagic acid was 60 times more powerful than quercetin. Consequently, these AR inhibitors are able to prevent the biochemical conversion of glucose to sorbitol in the polyol pathway and then reduce

Mus musculus (CF-1™ strain) were used by Da Silva et al. [27] to test the genotoxic and antigenotoxic potential of camu camu fruit juice after acute (single dose for one day), subacute (for 28 consecutive days), and chronic (for 56 consecutive days) oral administration. None of the fruit juice concentrations (25, 50, and 100%) tested exerted any genotoxic effect on blood cells in male and female mice. In the ex vivo test, with the alkaline comet assay, the fruit juice demonstrated antigenotoxic effect after acute, subacute, and chronic treatments. However, the acute administration of the fruit juice produced the lowest values in both damage index and damage frequency. The researchers associated the protective effect, against DNA damage caused by hydrogen peroxide, to the elevated levels of vitamin C, as well as to the flavonoids and phenolic compounds present in the fruit juice of camu camu; together these phytochemi-

Furthermore, several studies using microbial and animal models have demonstrated the antimutagenic properties of the camu camu fruits. In Peru, pioneering investigations were conducted by Gutiérrez [28], who tested the antimutagenic properties of an aqueous extract of fruit using in vitro and in vivo models. In the in vitro model, cultures of the CHO-K1 cell line from hamster (Cricetulus griseus) ovary were exposed to hydrogen peroxide and co-treated with fruit extract (concentrations at 1, 5, and 10%). The camu camu fruit extract had a significant capacity to protect against chromosomal aberrations induced by reactive oxygen species in a dosedependent manner. Also, in the in vivo model using fruit fly (Drosophila melanogaster) specimens, the antimutagenic activity of the fruit extract against the mutagenic effect induced by N-ethyl-Nnitrosourea (concentrations at 0.01 and 0.1 mmol) was demonstrated. This DNA damage protection effect was tested by the somatic mutation and recombination test that displayed a significant reduction in the frequency of wing spots (55.0–74.4%) in flies co-treated with 25% of fruit aqueous extract. In addition, Sánchez [57] demonstrated with the Ames test that a phenolic compound-rich fraction from the fruit pulp displayed antimutagenic activity (36.7–91.5%) in a

which was demonstrated in several studies [74–79].

3.4. DNA damage and cancer protection effects

cals are very able to eliminate free radicals.

diabetic complications [81–83].

An in vitro study based in the angiotensin converting enzyme-1 (ACE) inhibition displayed that the camu camu fruit pulp lacks antihypertension properties in the 10-mg/mL aqueous extract [26]. However, when spray-dried powders of fruit pulp were added (0.5–1.0%) to samples of lactic acid bacterial fermented soymilk, there was a higher ACE inhibitory effect, suggesting a synergic interaction between camu camu and soymilk active compounds.

#### 3.3. Antidiabetic activity

Some studies have shown that camu camu has antidiabetic activities, which may indicate its potential to treat this disease. For example, studies conducted by De Souza Schmidt Gonçalves et al. [56], De Azevêdo et al. [59], and Fujita et al. [26, 72] with fruit methanolic and polyamidepurified extracts, fruit depulping residue, pulp extract powders (spray drying and freezedrying), and a probiotic beverage from dried powder of fruit pulp combined with soymilk, respectively, have shown in vitro antidiabetic properties due to the combination of moderate alpha-amylase and potent alpha-glucosidase inhibitory activities. This antienzymatic association is considered appropriate as means of modulating carbohydrate digestion and retarding postprandial glycemia, which is an efficient strategy to manage early stages of diabetes type 2 [73]. Also, an in vivo assay with obese male R. norvegicus conducted by Nascimento et al. [44] treated with 25 mL of pulp for a duration of 4 weeks showed a significant reduction in both blood glucose (23%) and insulin activities (44.5%). The authors attribute the hypoglycemic activity to the large quantity of soluble fibers (Table 1) that form complexes with monosaccharides. Furthermore, studies with healthy young adults (20–35 years old) who received oral administration of pulp nectar [64] or encapsulated freeze-dried pulp displayed a significant hypoglycemic effects in 15 days [65, 66]. Finally, Balisteiro et al. [63] showed that healthy subjects that have ingested a polyphenols-rich juice of camu camu significantly reduced the blood glucose levels (31%) after a carbohydrate-rich meal ingestion. The hypoglycemic activity of these preparations can be attributed to polyphenols of the camu camu fruits. Polyphenols have two corroborated hypoglycemic mechanisms. The first is related to inhibitory action of the carbohydrate digestive enzymes alpha-amylase and alpha-glucosidase [26, 44, 56, 63, 72] because these inhibitory activities have high-positive correlations (Pearson's correlation coefficient ≥ 0.50) with the concentration of different phenolic compounds (casuarictin, ellagic acid, quercetin, syringic acid, myricetin, etc.) [26, 56]. Second, intestinal monosaccharides transporters such as sodiumdependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) were inhibited, which was demonstrated in several studies [74–79].

Additionally, Ueda et al. [80] isolated three aldose reductase (AR) inhibitors from an 80% methanolic leaf extract: ellagic acid, 4-O-methylellagic acid, and 4-(α-rhamnopyranosyl)ellagic acid. The last phenolic compound showed the strongest uncompetitive inhibition against human recombinant AR and rat lens AR. Also, the inhibitory activity of 4-(α-rhamnopyranosyl)ellagic acid was 60 times more powerful than quercetin. Consequently, these AR inhibitors are able to prevent the biochemical conversion of glucose to sorbitol in the polyol pathway and then reduce diabetic complications [81–83].

#### 3.4. DNA damage and cancer protection effects

excretion was increased. In addition, assays performed by Nascimento et al. [44] in a rat model of diet-induced obesity that ingested daily 25 mL of camu camu fruit pulp by 12 weeks resulted in a significant weights reduction of the fat in white adipose tissues. Triacylglycerols (↓40.6%), total cholesterol (↓39.6%), LDL cholesterol (↓2.14%), and VLDL cholesterol (↓36.4%) levels were also decreased, and HDL cholesterol (↑12.3%) increased in experimental groups. Similar experiments conducted by De Souza Schmidt Gonçalves et al. [61] in a type 1 diabetic rat model (streptozotocin induced) receiving oral administration of 1 or 3 g/kg by body weight of aqueous fruit pulp extract by 30 days, significantly reduced triacylglycerols and total cholesterol levels, and an increase in HDL cholesterol. Additionally, some controlled clinical trials with healthy young adults (20–35 years old) who received oral administration of pulp nectar [64] or encapsulated freeze-dried pulp for approximately 2 weeks [65, 66] displayed a significant improvement in their biochemical lipid profiles, due to hypolipidemic effects. The improvement in the biochemical profile of obesity is associated with an increase in lipid elimination in feces (50%) and by the liver (140%) due to the existence of dietary fiber in the fruit pulp [2, 41]. Dietary fibers have shown multiple beneficial health properties by their role in energy intake regulation and obesity development, which are related to its peculiar physicochemical characteristics. Some mechanisms suggested how dietary fiber aids in weight management include promoting satiation, extended signals of satiety, decreasing transport of macronutrients, and altering secretion of

An in vitro study based in the angiotensin converting enzyme-1 (ACE) inhibition displayed that the camu camu fruit pulp lacks antihypertension properties in the 10-mg/mL aqueous extract [26]. However, when spray-dried powders of fruit pulp were added (0.5–1.0%) to samples of lactic acid bacterial fermented soymilk, there was a higher ACE inhibitory effect,

Some studies have shown that camu camu has antidiabetic activities, which may indicate its potential to treat this disease. For example, studies conducted by De Souza Schmidt Gonçalves et al. [56], De Azevêdo et al. [59], and Fujita et al. [26, 72] with fruit methanolic and polyamidepurified extracts, fruit depulping residue, pulp extract powders (spray drying and freezedrying), and a probiotic beverage from dried powder of fruit pulp combined with soymilk, respectively, have shown in vitro antidiabetic properties due to the combination of moderate alpha-amylase and potent alpha-glucosidase inhibitory activities. This antienzymatic association is considered appropriate as means of modulating carbohydrate digestion and retarding postprandial glycemia, which is an efficient strategy to manage early stages of diabetes type 2 [73]. Also, an in vivo assay with obese male R. norvegicus conducted by Nascimento et al. [44] treated with 25 mL of pulp for a duration of 4 weeks showed a significant reduction in both blood glucose (23%) and insulin activities (44.5%). The authors attribute the hypoglycemic activity to the large quantity of soluble fibers (Table 1) that form complexes with monosaccharides. Furthermore, studies with healthy young adults (20–35 years old) who received oral administration of pulp nectar [64] or encapsulated freeze-dried pulp displayed a significant hypoglycemic effects in 15 days [65, 66]. Finally, Balisteiro et al. [63] showed that healthy subjects that have ingested a polyphenols-rich juice of camu camu significantly reduced the blood glucose levels

suggesting a synergic interaction between camu camu and soymilk active compounds.

gastrointestinal hormones [67–71].

96 Breeding and Health Benefits of Fruit and Nut Crops

3.3. Antidiabetic activity

Mus musculus (CF-1™ strain) were used by Da Silva et al. [27] to test the genotoxic and antigenotoxic potential of camu camu fruit juice after acute (single dose for one day), subacute (for 28 consecutive days), and chronic (for 56 consecutive days) oral administration. None of the fruit juice concentrations (25, 50, and 100%) tested exerted any genotoxic effect on blood cells in male and female mice. In the ex vivo test, with the alkaline comet assay, the fruit juice demonstrated antigenotoxic effect after acute, subacute, and chronic treatments. However, the acute administration of the fruit juice produced the lowest values in both damage index and damage frequency. The researchers associated the protective effect, against DNA damage caused by hydrogen peroxide, to the elevated levels of vitamin C, as well as to the flavonoids and phenolic compounds present in the fruit juice of camu camu; together these phytochemicals are very able to eliminate free radicals.

Furthermore, several studies using microbial and animal models have demonstrated the antimutagenic properties of the camu camu fruits. In Peru, pioneering investigations were conducted by Gutiérrez [28], who tested the antimutagenic properties of an aqueous extract of fruit using in vitro and in vivo models. In the in vitro model, cultures of the CHO-K1 cell line from hamster (Cricetulus griseus) ovary were exposed to hydrogen peroxide and co-treated with fruit extract (concentrations at 1, 5, and 10%). The camu camu fruit extract had a significant capacity to protect against chromosomal aberrations induced by reactive oxygen species in a dosedependent manner. Also, in the in vivo model using fruit fly (Drosophila melanogaster) specimens, the antimutagenic activity of the fruit extract against the mutagenic effect induced by N-ethyl-Nnitrosourea (concentrations at 0.01 and 0.1 mmol) was demonstrated. This DNA damage protection effect was tested by the somatic mutation and recombination test that displayed a significant reduction in the frequency of wing spots (55.0–74.4%) in flies co-treated with 25% of fruit aqueous extract. In addition, Sánchez [57] demonstrated with the Ames test that a phenolic compound-rich fraction from the fruit pulp displayed antimutagenic activity (36.7–91.5%) in a

dose-dependent manner against the mutagenic compound 3-amino-1,4-dimethyl-5H-pyrido[4,3 b]indole. Further, in the two investigations on the frequency of micronucleated polychromated erythrocytes of bone marrow cells of albine mice (M. musculus) induced with cyclophosphamide and co-treated with fruit pulp by orogastric gavage for 10–15 consecutive days, there was a significant and drastic (from 16 to 90%) reduction of micronucleous frequency in a dosedependent manner [21, 84]. Additionally, the cytoprotective activity of the camu camu fruit against the mutagenic damage caused by potassium bromate (KBrO3) was also demonstrated. Animals were treated by oral administration of a fruit aqueous extract (50 mg/kg) for 35 consecutive days. In the tenth day of treatment, albine mice were intraperitoneally injected with a solution of KBrO3 (dosage of 68.5 mg/kg) to induce mutagenic injury. After a meticulous comparative analysis of the DNA fragmentation degree, with the alkaline comet assay, a great inhibitory capability of the fruit pulp against the DNA-damaging effects of KBrO3 in blood, kidney, and liver cells was noticeable. This strong protective action was potentially attributed to the high content of antioxidants such as vitamin C and flavonoids present in the fruits of this fabulous Amazonian plant [85].

Malpighia glabra, Passiflora edulis, Punica granatum, Ribes nigrum, Vaccinium corymbosum, and V. oxycoccus). The liver of the rats was then injured by the injection of D-galactosamine (GalN). Only the juice of camu camu significantly suppressed the GalN-induced liver injury. This hepatoprotective activity was due to the active compound 1-methylmalate, which was isolated by bioassay-guided solvent fractionation and silica gel column chromatography of the camu camu fruit juice. To date, however, the molecular mechanism by which this active compound suppresses GalN-induced liver injury remains undiscovered. Similarly, the specific metabolic pathway and enzymes involved in the biosynthesis of 1-methymalate in camu camu are unknown. It is probable that the Krebs cycle provides malate for the biosynthesis of this active compound, and specific S-adenosyl-L-methionine-dependent methylation of carboxyl groups methyl transferase adds the methyl moiety in carbon 1 of malate, which remains undiscovered.

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The effect of hot air-dried residue of camu camu fruits (seeds, peel, and residual pulp) on the neuroprotective effects in experimentally induced neurodegeneration was evaluated in Caenorhabditis elegans transgenic models for Alzheimer's disease and Parkinson's disease [87]. Treatments with low molecular weight fraction of hot air-dried residue significantly extended the life span in C. elegans by 20% and delayed Aβ1–<sup>42</sup> aggregation induced paralysis by 21% in the Alzheimer's disease model. Additionally, in the 1-methyl-4-phenylpyridinium-induced oxidative dopaminergic neurotoxicity model for Parkinson's disease treatment with the same fraction of the fruit extract resulted in significant abrogation in neurotoxicity by 15–21%. These health-relevant effects were inferred mostly due to polar acidic low-molecular-weight bioac-

Recently, a Brazilian research group [88] evaluated the effect of the oral administration of the fruit powdered pulp extract in immunological parameters in Oreochromis niloticus (Nile tilapia). Fishes were fed for 5 weeks using various dosages (0–500 mg of camu camu extract/kg of feed). At the end of the trial period, fishes were inoculated in the swim bladder with the pathogenic bacteria Aeromonas hydrophila to induce an acute aerocystitis. The immunological parameters were then analyzed after 6, 24, and 48 h of the infection. Results revealed that fish supplemented with camu camu fruit extracts had significantly increased immunological responses by increasing the white blood cells counts and exudate (lymphocytes, monocytes, neutrophils, and thrombocytes), leukocyte respiratory burst, serum lysozyme activity, serum bactericidal activity, direct agglutination, and melanomacrophage centers count. Notably, an increase in fish growth after 5 weeks, especially,

Also in pre-clinical studies using R. norvegicus (Holtzmann strain) with oral administration of an aqueous extract of fruit pulp (5 and 10%) by 5 days, the authors displayed a greater immunostimulatory activity in a dose-dependent manner, after the treatments with the vegetable beverage [89]. This immune stimulant action was in two ways, first was by increasing the number of circulating mature lymphocytes (2 times) and the second mechanism was by stimulating the phagocytic activity of the reticulum endothelial system (in average 75.71%).

3.6. Neuroprotective and immunological effects

tive fractions.

at a dose of 500 mg/kg was detected.

Recent reports also demonstrated the anticancer properties of camu camu fruit juice. In the first research conducted by Carvalho-Silva et al. [21], the authors used an in vitro cytotoxicity and antiproliferative assays. In these tests, the HepG2, a liver hepatocellular carcinoma cell line, was grown for 72 hours in media containing increasing concentrations (0–25 mg/mL) of a tropical fruit juice mix that contained fruit pulp of camu camu at 5%. The results indicate that the tropical fruit juice mix provided anticancer activity against HepG2 cell line because the proliferation ratio was inhibited in a dose-dependent manner (from 10 to 80%, approximately). Also, there was no cytotoxic effect up to 150 mg/mL, suggesting that the anticancer effect was independent of cytotoxicity. These health-promoting actions were associated with the high levels of vitamin C and anthocyanins (i.e., cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-rhamnoside, etc.) present in the tropical plant beverages that together contribute to the great antioxidant capability, consequently decreasing the free radicals to the lowest level and then the risk of cancer. The second study conducted by Asmat and Benites [86] used an in vivo experimental model. In this study, one group of R. norvegicus (Albinus strain) was injected subcutaneously with 21 mg/kg of 1,2-dimethylhydrazine from week 2 to 22 to induce colorectal cancer and co-treated from week 1 to 32 with fruit extract (containing fruit pulp and peel) at a dosage of 4.37 g/kg. The control group also received treatment to develop colorectal cancer but received a standard diet and water ad libitum for 32 weeks. The fruit extract of camu camu interfered with the progression to histological alterations such as metaplasia. In contrast, the control group showed major structural damage, pseudostratification, necrosis, and high mitotic index. Once again, this demonstrated the multiple beneficial properties of camu camu fruits.

#### 3.5. Hepatoprotective activity

Akachi et al. [18] conducted experiments with R. norvegicus (Wistar strain), which were fed for 7 days with lyophilized fruit juice of camu camu or one of 11 other fruit juices (Averrhoa carambola, Citrus depressa, Hippophae rhamnoides, Hylocereus costaricensis, Litchi chinensis, Malpighia glabra, Passiflora edulis, Punica granatum, Ribes nigrum, Vaccinium corymbosum, and V. oxycoccus). The liver of the rats was then injured by the injection of D-galactosamine (GalN). Only the juice of camu camu significantly suppressed the GalN-induced liver injury. This hepatoprotective activity was due to the active compound 1-methylmalate, which was isolated by bioassay-guided solvent fractionation and silica gel column chromatography of the camu camu fruit juice. To date, however, the molecular mechanism by which this active compound suppresses GalN-induced liver injury remains undiscovered. Similarly, the specific metabolic pathway and enzymes involved in the biosynthesis of 1-methymalate in camu camu are unknown. It is probable that the Krebs cycle provides malate for the biosynthesis of this active compound, and specific S-adenosyl-L-methionine-dependent methylation of carboxyl groups methyl transferase adds the methyl moiety in carbon 1 of malate, which remains undiscovered.

#### 3.6. Neuroprotective and immunological effects

dose-dependent manner against the mutagenic compound 3-amino-1,4-dimethyl-5H-pyrido[4,3 b]indole. Further, in the two investigations on the frequency of micronucleated polychromated erythrocytes of bone marrow cells of albine mice (M. musculus) induced with cyclophosphamide and co-treated with fruit pulp by orogastric gavage for 10–15 consecutive days, there was a significant and drastic (from 16 to 90%) reduction of micronucleous frequency in a dosedependent manner [21, 84]. Additionally, the cytoprotective activity of the camu camu fruit against the mutagenic damage caused by potassium bromate (KBrO3) was also demonstrated. Animals were treated by oral administration of a fruit aqueous extract (50 mg/kg) for 35 consecutive days. In the tenth day of treatment, albine mice were intraperitoneally injected with a solution of KBrO3 (dosage of 68.5 mg/kg) to induce mutagenic injury. After a meticulous comparative analysis of the DNA fragmentation degree, with the alkaline comet assay, a great inhibitory capability of the fruit pulp against the DNA-damaging effects of KBrO3 in blood, kidney, and liver cells was noticeable. This strong protective action was potentially attributed to the high content of antioxidants such as vitamin C and flavonoids present in the fruits of this

Recent reports also demonstrated the anticancer properties of camu camu fruit juice. In the first research conducted by Carvalho-Silva et al. [21], the authors used an in vitro cytotoxicity and antiproliferative assays. In these tests, the HepG2, a liver hepatocellular carcinoma cell line, was grown for 72 hours in media containing increasing concentrations (0–25 mg/mL) of a tropical fruit juice mix that contained fruit pulp of camu camu at 5%. The results indicate that the tropical fruit juice mix provided anticancer activity against HepG2 cell line because the proliferation ratio was inhibited in a dose-dependent manner (from 10 to 80%, approximately). Also, there was no cytotoxic effect up to 150 mg/mL, suggesting that the anticancer effect was independent of cytotoxicity. These health-promoting actions were associated with the high levels of vitamin C and anthocyanins (i.e., cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-rhamnoside, etc.) present in the tropical plant beverages that together contribute to the great antioxidant capability, consequently decreasing the free radicals to the lowest level and then the risk of cancer. The second study conducted by Asmat and Benites [86] used an in vivo experimental model. In this study, one group of R. norvegicus (Albinus strain) was injected subcutaneously with 21 mg/kg of 1,2-dimethylhydrazine from week 2 to 22 to induce colorectal cancer and co-treated from week 1 to 32 with fruit extract (containing fruit pulp and peel) at a dosage of 4.37 g/kg. The control group also received treatment to develop colorectal cancer but received a standard diet and water ad libitum for 32 weeks. The fruit extract of camu camu interfered with the progression to histological alterations such as metaplasia. In contrast, the control group showed major structural damage, pseudostratification, necrosis, and high mitotic index. Once again, this demonstrated

Akachi et al. [18] conducted experiments with R. norvegicus (Wistar strain), which were fed for 7 days with lyophilized fruit juice of camu camu or one of 11 other fruit juices (Averrhoa carambola, Citrus depressa, Hippophae rhamnoides, Hylocereus costaricensis, Litchi chinensis,

fabulous Amazonian plant [85].

98 Breeding and Health Benefits of Fruit and Nut Crops

the multiple beneficial properties of camu camu fruits.

3.5. Hepatoprotective activity

The effect of hot air-dried residue of camu camu fruits (seeds, peel, and residual pulp) on the neuroprotective effects in experimentally induced neurodegeneration was evaluated in Caenorhabditis elegans transgenic models for Alzheimer's disease and Parkinson's disease [87]. Treatments with low molecular weight fraction of hot air-dried residue significantly extended the life span in C. elegans by 20% and delayed Aβ1–<sup>42</sup> aggregation induced paralysis by 21% in the Alzheimer's disease model. Additionally, in the 1-methyl-4-phenylpyridinium-induced oxidative dopaminergic neurotoxicity model for Parkinson's disease treatment with the same fraction of the fruit extract resulted in significant abrogation in neurotoxicity by 15–21%. These health-relevant effects were inferred mostly due to polar acidic low-molecular-weight bioactive fractions.

Recently, a Brazilian research group [88] evaluated the effect of the oral administration of the fruit powdered pulp extract in immunological parameters in Oreochromis niloticus (Nile tilapia). Fishes were fed for 5 weeks using various dosages (0–500 mg of camu camu extract/kg of feed). At the end of the trial period, fishes were inoculated in the swim bladder with the pathogenic bacteria Aeromonas hydrophila to induce an acute aerocystitis. The immunological parameters were then analyzed after 6, 24, and 48 h of the infection. Results revealed that fish supplemented with camu camu fruit extracts had significantly increased immunological responses by increasing the white blood cells counts and exudate (lymphocytes, monocytes, neutrophils, and thrombocytes), leukocyte respiratory burst, serum lysozyme activity, serum bactericidal activity, direct agglutination, and melanomacrophage centers count. Notably, an increase in fish growth after 5 weeks, especially, at a dose of 500 mg/kg was detected.

Also in pre-clinical studies using R. norvegicus (Holtzmann strain) with oral administration of an aqueous extract of fruit pulp (5 and 10%) by 5 days, the authors displayed a greater immunostimulatory activity in a dose-dependent manner, after the treatments with the vegetable beverage [89]. This immune stimulant action was in two ways, first was by increasing the number of circulating mature lymphocytes (2 times) and the second mechanism was by stimulating the phagocytic activity of the reticulum endothelial system (in average 75.71%).

#### 3.7. Antibacterial and antiparasitic activities

Recently, several research groups have investigated the antibacterial and antiparasitic activities of botanical extracts from camu camu. With respect to antibacterial activity, the first report was from a Japanese research group (Myoda et al. [19]) who showed that the methanolic extracts (100% methanol) from fruit peel and seeds have strong antimicrobial activity against Staphylococcus aureus at 5 mg/mL, suggesting that lipophylic chemicals are responsible for the selective antistaphylococcal activity. Also, a Peruvian research group reported the antistaphylococcal activity of hydroalcoholic extracts (70% ethanol) derived from leaves and bark [90]. Again, Brazilian researchers recorded antistaphylococcal activity of the lyophilized pulp, spouted bed dried pulp, and spray-dried pulp with minimum inhibitory concentration (MIC) of extracts of 0.08 mg/mL [26, 91]. Similarly, another Brazilian research group showed the antistaphylococcal activity of the fruit industrial residue (seeds and peel). Both fresh, freeze dried, and hot air dried residues showed higher inhibition zones (>10 mm) and lower MIC (0.31–2.50 mg/mL) against S. aureus. In addition, the polyphenolic-rich fractions provided these antibacterial activities with inhibition zones from 13.1 to 16.1 mm and MIC value of 2.5 mg/mL. Furthermore, another Peruvian research group demonstrated that methanolic extracts from pulp and seeds possess higher antibacterial effects against the cariogenic bacteria Streptococcus mutans and S. sanguinis. The MIC of the pulp extract showed a range of 50–75 μg/mL; however, the seed antibacterial activity was detected at very low levels [92]. Finally, recently another Japanese research group isolated four polyphenolic antimicrobial constituents (acylphloroglucinols) from the n-hexane extracts of peel and seeds, these compounds were Isomyrtucommulone B, Myrciarone A, Myrciarone B, and Rhodomyrtone. The second and third compounds were confirmed to be new acylphloroglucinols [20]. The four compounds showed antimicrobial activities against Gram-positive bacteria such as Bacillus subtilis, B. cereus, Micrococcus luteus, S. aureus, S. epidermidis, and S. mutans but were inactive against Gram-negative bacteria and fungi. Several investigations at transcriptomic, proteomic, and metabolomic levels have revealed the molecular mechanism involved in the antimicrobial activity of Rhodomyrtone against Gram-positive bacteria [93–96].With respect to antiparasitic activities, researchers from England [51, 97] reported that a mixture of 10 myricetin and quercetin glycosides isolated from the aqueous acetic acid–soluble fraction of methanolic extracts of camu camu were potent inhibitors of the GSH-haemin reaction. Also, the aqueous ethanolic extract (70%) of fruit peel, leaves, and seeds exhibited antiplasmodial activity with the ferriprotoporphyrin inhibition test with IC50 < 5.0 μg/mL [50]. Additionally, the aqueous and the ethanolic (70%) extracts from the bark displayed inhibitory activity against the Plasmodium falciparum strain FCR3 (chloroquine resistant) with IC50 values of 3 and 6 μg/mL, respectively [98]. In addition, the dichloromethanolic extract of camu camu leaves exhibited a significant in vitro growth inhibition of P. falciparum and Leishmania amazonensis with IC50 values of 1.05 and 6.41 μg/mL, respectively [99].

that camu camu fruits extract significantly increased the daily sperm production, stages IX and XI of mitosis and stage XII of meiosis. In combination with black maca, spermiation stages, mitosis, and meiosis were increased. The authors concluded that camu camu fruits potentially improve spermatogenesis and mixing with black maca tubers increased the stages of mitosis, meiosis, and spermiation of the spermatogenic cycle as assayed by the transillumination

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Additionally, two in vitro investigations demonstrated that the hydroalcoholic and aqueous fruit extracts (containing peel, pulp, and seeds) of camu camu could be added in cosmetic formulations, since fruit extracts empower the sun protection factor against UVB radiation. This peculiar characteristic is attributable to the high levels of vitamin C and phenolic com-

Finally, a research of Yuyama et al. [103] demonstrated the beneficial impacts of mixing fruits pulp of camu camu and Euterpe oleracea to treat preschool children (2–6 years old) with anemia

Recently, our research group used a total of 24,551,882 high-quality reads to assemble the fruit (unripe, semiripe, and ripe) transcriptome of camu camu. In total 70,048 unigenes were obtained in the meta-assembly (mean length of 1150 bp and N50 = 1775 bp). These unigenes were annotated by searching homologous sequences in multiple databases (i.e., NCBI nonredundant (nr), UniProtKB, TAIR, GR\_protein, FB, MGI, etc.). The top three plant species that contributed the greatest number of gene annotations were Vitis vinifera, Theobroma cacao, and Populus trichocarpa. Of the three core GO annotation categories, biological processes comprised 53.6% of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1%, respectively. In total, 160 metabolic pathways were

Based on the fruit transcriptome analysis, five metabolic pathways for vitamin C biosynthesis [48] were reconstructed: animal-like pathway, myo-inositol pathway, L-gulose pathway, Dmannose/L-galactose pathway, and uronic acid pathway. Gene coding enzymes involved in the ascorbate-glutathione cycle were also identified (Figure 5). From these pathways, the Dmannose/L-galactose pathway is the best characterized in several plant species [104–106]. This pathway involves the sequential enzymatic conversion of D-mannose-1-phosphate in to Vitamin C. These enzymatic reactions are as follows: GDP-D-mannose synthesis from D-mannose-1-phosphate and GTP is catalyzed by GDP-D-mannose pyrophosphorylase (E.C. 2.7.7.13), and then, GDP-D-mannose is converted to GDP-L-galactose by a reversible double epimerization,


technique [100].

pounds in the fruit [101, 102].

and chronic malnutrition.

reconstructed [48].

catalyzed by GDP-mannose-3<sup>0</sup>

4. Functional genomic characteristics

4.1. Transcriptome de novo asembly and annotation

4.2. Metabolic pathways for vitamin C biosynthesis

,50

#### 3.8. Other bioactivities

Animal experiments using R. norvegicus to test the effect of a fruit extract of camu camu alone and in combination with a powdered tuber extract of black maca (Lepidium meyenii) showed that camu camu fruits extract significantly increased the daily sperm production, stages IX and XI of mitosis and stage XII of meiosis. In combination with black maca, spermiation stages, mitosis, and meiosis were increased. The authors concluded that camu camu fruits potentially improve spermatogenesis and mixing with black maca tubers increased the stages of mitosis, meiosis, and spermiation of the spermatogenic cycle as assayed by the transillumination technique [100].

Additionally, two in vitro investigations demonstrated that the hydroalcoholic and aqueous fruit extracts (containing peel, pulp, and seeds) of camu camu could be added in cosmetic formulations, since fruit extracts empower the sun protection factor against UVB radiation. This peculiar characteristic is attributable to the high levels of vitamin C and phenolic compounds in the fruit [101, 102].

Finally, a research of Yuyama et al. [103] demonstrated the beneficial impacts of mixing fruits pulp of camu camu and Euterpe oleracea to treat preschool children (2–6 years old) with anemia and chronic malnutrition.

#### 4. Functional genomic characteristics

3.7. Antibacterial and antiparasitic activities

100 Breeding and Health Benefits of Fruit and Nut Crops

and 6.41 μg/mL, respectively [99].

3.8. Other bioactivities

Recently, several research groups have investigated the antibacterial and antiparasitic activities of botanical extracts from camu camu. With respect to antibacterial activity, the first report was from a Japanese research group (Myoda et al. [19]) who showed that the methanolic extracts (100% methanol) from fruit peel and seeds have strong antimicrobial activity against Staphylococcus aureus at 5 mg/mL, suggesting that lipophylic chemicals are responsible for the selective antistaphylococcal activity. Also, a Peruvian research group reported the antistaphylococcal activity of hydroalcoholic extracts (70% ethanol) derived from leaves and bark [90]. Again, Brazilian researchers recorded antistaphylococcal activity of the lyophilized pulp, spouted bed dried pulp, and spray-dried pulp with minimum inhibitory concentration (MIC) of extracts of 0.08 mg/mL [26, 91]. Similarly, another Brazilian research group showed the antistaphylococcal activity of the fruit industrial residue (seeds and peel). Both fresh, freeze dried, and hot air dried residues showed higher inhibition zones (>10 mm) and lower MIC (0.31–2.50 mg/mL) against S. aureus. In addition, the polyphenolic-rich fractions provided these antibacterial activities with inhibition zones from 13.1 to 16.1 mm and MIC value of 2.5 mg/mL. Furthermore, another Peruvian research group demonstrated that methanolic extracts from pulp and seeds possess higher antibacterial effects against the cariogenic bacteria Streptococcus mutans and S. sanguinis. The MIC of the pulp extract showed a range of 50–75 μg/mL; however, the seed antibacterial activity was detected at very low levels [92]. Finally, recently another Japanese research group isolated four polyphenolic antimicrobial constituents (acylphloroglucinols) from the n-hexane extracts of peel and seeds, these compounds were Isomyrtucommulone B, Myrciarone A, Myrciarone B, and Rhodomyrtone. The second and third compounds were confirmed to be new acylphloroglucinols [20]. The four compounds showed antimicrobial activities against Gram-positive bacteria such as Bacillus subtilis, B. cereus, Micrococcus luteus, S. aureus, S. epidermidis, and S. mutans but were inactive against Gram-negative bacteria and fungi. Several investigations at transcriptomic, proteomic, and metabolomic levels have revealed the molecular mechanism involved in the antimicrobial activity of Rhodomyrtone against Gram-positive bacteria [93–96].With respect to antiparasitic activities, researchers from England [51, 97] reported that a mixture of 10 myricetin and quercetin glycosides isolated from the aqueous acetic acid–soluble fraction of methanolic extracts of camu camu were potent inhibitors of the GSH-haemin reaction. Also, the aqueous ethanolic extract (70%) of fruit peel, leaves, and seeds exhibited antiplasmodial activity with the ferriprotoporphyrin inhibition test with IC50 < 5.0 μg/mL [50]. Additionally, the aqueous and the ethanolic (70%) extracts from the bark displayed inhibitory activity against the Plasmodium falciparum strain FCR3 (chloroquine resistant) with IC50 values of 3 and 6 μg/mL, respectively [98]. In addition, the dichloromethanolic extract of camu camu leaves exhibited a significant in vitro growth inhibition of P. falciparum and Leishmania amazonensis with IC50 values of 1.05

Animal experiments using R. norvegicus to test the effect of a fruit extract of camu camu alone and in combination with a powdered tuber extract of black maca (Lepidium meyenii) showed

#### 4.1. Transcriptome de novo asembly and annotation

Recently, our research group used a total of 24,551,882 high-quality reads to assemble the fruit (unripe, semiripe, and ripe) transcriptome of camu camu. In total 70,048 unigenes were obtained in the meta-assembly (mean length of 1150 bp and N50 = 1775 bp). These unigenes were annotated by searching homologous sequences in multiple databases (i.e., NCBI nonredundant (nr), UniProtKB, TAIR, GR\_protein, FB, MGI, etc.). The top three plant species that contributed the greatest number of gene annotations were Vitis vinifera, Theobroma cacao, and Populus trichocarpa. Of the three core GO annotation categories, biological processes comprised 53.6% of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1%, respectively. In total, 160 metabolic pathways were reconstructed [48].

#### 4.2. Metabolic pathways for vitamin C biosynthesis

Based on the fruit transcriptome analysis, five metabolic pathways for vitamin C biosynthesis [48] were reconstructed: animal-like pathway, myo-inositol pathway, L-gulose pathway, Dmannose/L-galactose pathway, and uronic acid pathway. Gene coding enzymes involved in the ascorbate-glutathione cycle were also identified (Figure 5). From these pathways, the Dmannose/L-galactose pathway is the best characterized in several plant species [104–106]. This pathway involves the sequential enzymatic conversion of D-mannose-1-phosphate in to Vitamin C. These enzymatic reactions are as follows: GDP-D-mannose synthesis from D-mannose-1-phosphate and GTP is catalyzed by GDP-D-mannose pyrophosphorylase (E.C. 2.7.7.13), and then, GDP-D-mannose is converted to GDP-L-galactose by a reversible double epimerization, catalyzed by GDP-mannose-3<sup>0</sup> ,50 -epimerase (E.C. 5.1.3.18); further, GDP-L-galactose is

transformed by GDP-L-galactose:hexose-1-phosphate guanyltransferase (E.C. 2.7.7.69) to Lgalactose-1-phosphate, which is subsequently hydrolyzed to L-galactose and inorganic phosphate by L-galactose-1-phosphate phosphatase (E.C. 3.1.3.25). L-galactose is next oxidized to L-galactono-1,4-lactone by the NAD-dependent L-galactose dehydrogenase (E.C. 1.1.1.316), and finally, L-galactono-1,4-lactone is oxidized to vitamin C by L-galactono-1,4-lactone dehy-

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4.3. Metabolic pathways involved in health-promoting phytochemicals biosynthesis

As previously mentioned, most of the health-promoting phytochemical compounds identified in camu camu are specialized metabolites, commonly known as secondary metabolites. Biosynthesis of these structural diverse molecules starts from key basic pathways, for instance the Embden-Meyerhof-Parnas pathway (also known as the glycolysis), pentose phosphate pathway, and the Shikimate pathway. The latter pathway produces chorismate, a common precursor for the tryptophan pathway, the phenylalanine/tyrosine pathways, and the metabolic pathways for the biosynthesis of folate, salicylate, and phylloquinone [107]. Subsequently, these three aromatic amino acids are used as biosynthetic precursors in several metabolic pathways to produce a diverse array of secondary metabolites (i.e., terpenoids, phenolic compounds such as flavonols, anthocyanins, ellagic acid and derivatives, ellagitannins, gallic acid and derivatives, etc.), depending on several biological and environmental factors [108]. From the annotated fruit transcriptome of camu camu, we were able to reconstruct more than 160 metabolic pathways [48]. These include several pathways involved directly in secondary metabolite biosynthesis, for example, the anthocyanins, carotenoids, flavonoids, phenylpropanoids, and terpenoids biosynthesis pathways. The universal biosynthetic precursor (chorismate) for all these pathways is synthesized in the Shikimate pathway (Figure 6). In this pathway, seven enzymatic reactions biochemically transform phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (metabolic intermediates in glycolysis and the pentose phosphate pathways, respectively) to chorismate. The first committed step of the shikimate pathway is an aldol condensation of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to produce 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), this reaction is catalyzed by DAHP synthase (E.C. 2.5.1.54). Further, 3-Dehydroquinate synthase (E.C. 4.2.3.4) converts DAHP to 3-dehydroquinate using a divalent cation (i.e., Co2+) and NAD+ cofactors via five consecutive chemical reactions: alcohol oxidation, β-elimination of inorganic phosphate, carbonyl reduction, ring opening, and intramolecular aldol condensation. The third enzymatic reaction catalyzed by 3-dehydroquinate dehydratase (E.C. 4.2.1.10) includes the dehydration of 3-dehydroquinate to 3-dehydroshikimate to introduce the first double bond in the ring, and the fourth reaction catalyzed by shikimate:NADP<sup>+</sup> oxidoreductase (E.C. 1.1.1.25) is a reversible reduction of 3-dehydroshikimate into shikimate using NADPH. The fifth enzyme (shikimate kinase [2.7.1.71]) catalyzes the phosphorylation of the C3 hydroxyl group of shikimate using ATP as inorganic phosphate donor to yield shikimate-3-phosphate. Then, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C. 2.5.1.19) catalyzes the formation of EPSP, by transferring the enolpyruvyl moiety of PEP to the 5-hydroxyl position of shikimate-3 phosphate. Finally, chorismate synthase (E.C. E.C. 4.2.3.5), the last enzyme of the shikimate

drogenase (E.C. 1.3.2.3).

Figure 5. Vitamin C biosynthesis and recycling pathways reconstructed from the fruit transcriptome database of camu camu. Source: Castro et al. [48].

transformed by GDP-L-galactose:hexose-1-phosphate guanyltransferase (E.C. 2.7.7.69) to Lgalactose-1-phosphate, which is subsequently hydrolyzed to L-galactose and inorganic phosphate by L-galactose-1-phosphate phosphatase (E.C. 3.1.3.25). L-galactose is next oxidized to L-galactono-1,4-lactone by the NAD-dependent L-galactose dehydrogenase (E.C. 1.1.1.316), and finally, L-galactono-1,4-lactone is oxidized to vitamin C by L-galactono-1,4-lactone dehydrogenase (E.C. 1.3.2.3).

#### 4.3. Metabolic pathways involved in health-promoting phytochemicals biosynthesis

As previously mentioned, most of the health-promoting phytochemical compounds identified in camu camu are specialized metabolites, commonly known as secondary metabolites. Biosynthesis of these structural diverse molecules starts from key basic pathways, for instance the Embden-Meyerhof-Parnas pathway (also known as the glycolysis), pentose phosphate pathway, and the Shikimate pathway. The latter pathway produces chorismate, a common precursor for the tryptophan pathway, the phenylalanine/tyrosine pathways, and the metabolic pathways for the biosynthesis of folate, salicylate, and phylloquinone [107]. Subsequently, these three aromatic amino acids are used as biosynthetic precursors in several metabolic pathways to produce a diverse array of secondary metabolites (i.e., terpenoids, phenolic compounds such as flavonols, anthocyanins, ellagic acid and derivatives, ellagitannins, gallic acid and derivatives, etc.), depending on several biological and environmental factors [108]. From the annotated fruit transcriptome of camu camu, we were able to reconstruct more than 160 metabolic pathways [48]. These include several pathways involved directly in secondary metabolite biosynthesis, for example, the anthocyanins, carotenoids, flavonoids, phenylpropanoids, and terpenoids biosynthesis pathways. The universal biosynthetic precursor (chorismate) for all these pathways is synthesized in the Shikimate pathway (Figure 6). In this pathway, seven enzymatic reactions biochemically transform phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (metabolic intermediates in glycolysis and the pentose phosphate pathways, respectively) to chorismate. The first committed step of the shikimate pathway is an aldol condensation of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to produce 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), this reaction is catalyzed by DAHP synthase (E.C. 2.5.1.54). Further, 3-Dehydroquinate synthase (E.C. 4.2.3.4) converts DAHP to 3-dehydroquinate using a divalent cation (i.e., Co2+) and NAD+ cofactors via five consecutive chemical reactions: alcohol oxidation, β-elimination of inorganic phosphate, carbonyl reduction, ring opening, and intramolecular aldol condensation. The third enzymatic reaction catalyzed by 3-dehydroquinate dehydratase (E.C. 4.2.1.10) includes the dehydration of 3-dehydroquinate to 3-dehydroshikimate to introduce the first double bond in the ring, and the fourth reaction catalyzed by shikimate:NADP<sup>+</sup> oxidoreductase (E.C. 1.1.1.25) is a reversible reduction of 3-dehydroshikimate into shikimate using NADPH. The fifth enzyme (shikimate kinase [2.7.1.71]) catalyzes the phosphorylation of the C3 hydroxyl group of shikimate using ATP as inorganic phosphate donor to yield shikimate-3-phosphate. Then, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C. 2.5.1.19) catalyzes the formation of EPSP, by transferring the enolpyruvyl moiety of PEP to the 5-hydroxyl position of shikimate-3 phosphate. Finally, chorismate synthase (E.C. E.C. 4.2.3.5), the last enzyme of the shikimate

Figure 5. Vitamin C biosynthesis and recycling pathways reconstructed from the fruit transcriptome database of camu

camu. Source: Castro et al. [48].

102 Breeding and Health Benefits of Fruit and Nut Crops

pathway, is itself biochemically unique in nature and catalyzes a 1,4-antielimination of the 3-phosphate group and C6-pro-R hydrogen from EPSP, introduces the second double bond in

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105

Some Peruvian research Institutions such as the National Institute of Agricultural Innovation (INIA), the Research Institute from the Peruvian Amazon (IIAP), and the Veterinary Institute for Tropical and High Altitude Research (IVITA), as well as Brazilian research Institutions such as the National Institute of Amazonian Research (INPA) and the Brazilian Agricultural Research Corporation (EMBRAPA), have implemented programs for ex situ conservation of camu camu. These genetic conservation programs involve the establishment of germplasm banks composed by accessions of botanical material collected from wild populations. In Peru, germplasm banks were established about 37 years ago [109] from seeds (due to the lack of vegetative propagation techniques) obtained from only 40% of the wild populations in the Loreto region (Imán S., personal communication, September 15, 2017). Consequently, further prospecting and collecting of botanical samples need to be carried at regional and distribution wide levels to maximize greatest genetic diversity. The efficiency of increasing banked accessions could be improved by incorporating vegetative propagation techniques. The two most plausible alternatives are improved grafting and root cuttings techniques, developed and refined by Peruvian and Brazilian researchers

The domestication process of camu camu was promoted by INIA and IVITA at the beginning of the 1990s with the installation of seven demonstration parcels in the community of Santa Ana, which are located in the Amazon River 30 km from Iquitos [109]. Further, since the beginning of the twenty-first century, INIA in association with IIAP implemented a genetic improvement program using an active community participation strategy and conventional plant breeding methods, based on Mendelian principles of inheritance [109]. This improvement program was focused on an ideotype characterized by precocity of fructification (beginning with the third year after germination, but with ≥0.5 kg of fruits per plant), high vitamin C content in fruit pulp (≥ 2.0 g per 100 g of fruit pulp), and larger fruits (fresh weight ≥ 10 g). The promoters of these programs touted that the first generation of genetically superior plants would be ready by 2010 and superior homozygous lines by 2016. Thus far, none of these goals

To overcome these drawbacks, a radical redefinition of ideotypes is necessary. Our current knowledge affords us the opportunity to create comprehensive ideotypes that is built upon detailed knowledge of plant genetics, biochemistry, physiology, anatomy, morphology, phenology, and ecology [113]. Additionally, including the state of the art technologies for multiomic data analysis (i.e., genomic, epigenomic, transcriptomic, proteomic, metabolomic, phenomic, etc.) will enable the rational design and application of innovative strategies for the domestication

5. Domestication strategy and efforts for genetic improvement

the ring to produce chorismate [107].

[110–112].

have been achieved.

Figure 6. The Shikimate pathway reconstructed from the fruit transcriptome database of camu camu.

pathway, is itself biochemically unique in nature and catalyzes a 1,4-antielimination of the 3-phosphate group and C6-pro-R hydrogen from EPSP, introduces the second double bond in the ring to produce chorismate [107].

#### 5. Domestication strategy and efforts for genetic improvement

Some Peruvian research Institutions such as the National Institute of Agricultural Innovation (INIA), the Research Institute from the Peruvian Amazon (IIAP), and the Veterinary Institute for Tropical and High Altitude Research (IVITA), as well as Brazilian research Institutions such as the National Institute of Amazonian Research (INPA) and the Brazilian Agricultural Research Corporation (EMBRAPA), have implemented programs for ex situ conservation of camu camu. These genetic conservation programs involve the establishment of germplasm banks composed by accessions of botanical material collected from wild populations. In Peru, germplasm banks were established about 37 years ago [109] from seeds (due to the lack of vegetative propagation techniques) obtained from only 40% of the wild populations in the Loreto region (Imán S., personal communication, September 15, 2017). Consequently, further prospecting and collecting of botanical samples need to be carried at regional and distribution wide levels to maximize greatest genetic diversity. The efficiency of increasing banked accessions could be improved by incorporating vegetative propagation techniques. The two most plausible alternatives are improved grafting and root cuttings techniques, developed and refined by Peruvian and Brazilian researchers [110–112].

The domestication process of camu camu was promoted by INIA and IVITA at the beginning of the 1990s with the installation of seven demonstration parcels in the community of Santa Ana, which are located in the Amazon River 30 km from Iquitos [109]. Further, since the beginning of the twenty-first century, INIA in association with IIAP implemented a genetic improvement program using an active community participation strategy and conventional plant breeding methods, based on Mendelian principles of inheritance [109]. This improvement program was focused on an ideotype characterized by precocity of fructification (beginning with the third year after germination, but with ≥0.5 kg of fruits per plant), high vitamin C content in fruit pulp (≥ 2.0 g per 100 g of fruit pulp), and larger fruits (fresh weight ≥ 10 g). The promoters of these programs touted that the first generation of genetically superior plants would be ready by 2010 and superior homozygous lines by 2016. Thus far, none of these goals have been achieved.

To overcome these drawbacks, a radical redefinition of ideotypes is necessary. Our current knowledge affords us the opportunity to create comprehensive ideotypes that is built upon detailed knowledge of plant genetics, biochemistry, physiology, anatomy, morphology, phenology, and ecology [113]. Additionally, including the state of the art technologies for multiomic data analysis (i.e., genomic, epigenomic, transcriptomic, proteomic, metabolomic, phenomic, etc.) will enable the rational design and application of innovative strategies for the domestication

Figure 6. The Shikimate pathway reconstructed from the fruit transcriptome database of camu camu.

104 Breeding and Health Benefits of Fruit and Nut Crops

and the genetic improvement program for camu camu. For example, using genome editing tools such as clustered regularly interspaced short palindromic repeats/associated protein-9 nuclease [CRISPR/Cas9 system], transcription activator-like effector nucleases [TALENs], and zinc finger nucleases [ZFNs] could be the molecular tools of choice to achieve the desired ideotypes [114–118], after obtaining the complete genome sequence of camu camu.

Author details

Juan C. Castro<sup>1</sup>

Perú

History, Chicago, IL, USA

(INIA), Iquitos, Perú

Tempore; 1996. p. 95

de biología; 1999. p. 8

Information Service. 1993;54:16-17

Genetics & Genomes. 2012;8(3):463-508

References

\*, J. Dylan Maddox2,3, Marianela Cobos4 and Sixto A. Imán<sup>5</sup>

*Myrciaria dubia* "Camu Camu" Fruit: Health-Promoting Phytochemicals and Functional Genomic Characteristics

http://dx.doi.org/10.5772/intechopen.73213

107

1 Specialized Unit of Biotechnology, Research Center of Natural Resources of the Amazon

3 Environmental Sciences, American Public University System, Charles Town, WV, USA

2 Pritzker Laboratory for Molecular Systematics and Evolution, The Field Museum of Natural

4 Laboratory of Biotechnology and Bioenergetics, Scientific University of Peru (UCP), Iquitos,

5 Conservation Area of Plant Genetic Resources, National Institute of Agricultural Innovation

[1] Lim TK. Myrciaria dubia. In: Edible medicinal and non medicinal plants. Springer

[2] Villachica H. El cultivo del camu camu (Myrciaria dubia H.B.K. McVaugh) en la Amazonía Peruana. Lima, Perú: Tratado de Cooperación Amazónica (TCA), Secretaría Pro-

[3] Alvarado E, Gutiérrez J, Benites E, Nomberto C. Número cromosómico de Myrciaria dubia (H.B.K.) McVaugh "camu camu". Lima, Perú: En primer congreso internacional

[4] Uchiyama H, Koyama T. Chromosomes of Myrciaria dubia, Myrtaceae. Chromosomo

[5] Grattapaglia D, Vaillancourt RE, Shepherd M, Thumma BR, Foley W, Külheim C, et al. Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus. Tree

[6] Da Costa IR, Dornelas MC, Forni-Martins ER. Nuclear genome size variation in fleshyfruited Neotropical Myrtaceae. Plant Systematics and Evolution. 2008;276(3–4):209-217

[7] Teixeira AS, Chaves L da S, Yuyama K. Esterases for examining the population structure of camu-camu (Myrciaria dubia (Kunth) McVaugh-Myrtaceae). Acta Amaz. 2004;34(1):75-88

(CIRNA), National University of the Peruvian Amazon (UNAP), Iquitos, Perú

\*Address all correspondence to: juan.castro@unapiquitos.edu.pe

Netherlands; (volume 3, Fruits); 2012. pp. 631-638

To accelerate the domestication and genetic improvement program to obtain de novo elite commercial varieties, the five-step strategy of genomics-based plant germplasm research recommended by Jia et al. [119] should be implemented: (1) the detection of genomic diversity in germplasm banks, (2) the conservation and protection of germplasm based on the knowledge of genomic diversity, (3) the use of diversity information to design a representative core collection, (4) the enhancement of germplasm banks using core collections, and (5) the discovery of new alleles and/or genes in the core collections.

To date, our research team has generated fruit transcriptome data and identified several of the genes involved in vitamin C biosynthesis that have proved to be polymorphic. For example, the D-mannose/L-galactose pathway mannose-1-phosphate guanylyltransferase (E.C. 2.7.7.13) contained >20 SNPs, GDP-mannose-3<sup>0</sup> ,50 -epimerase (E.C. 5.1.3.18) had 13 SNPs, whereas L-galactono-1,4-lactone dehydrogenase (E.C. 1.3.2.3) only had 5 SNPs. The animal-like pathway UTP:glucose-1-phosphate uridylyltransferase (E.C. 2.7.7.9) contained 7 SNPs. In the uronic acid pathway pectin esterase (E.C. 3.1.1.11) and galacturan-1,4-alpha-galacturonidase (E.C. 3.2.1.15) showed more than 20 and 14 SNPs, respectively. Finally, in the ascorbateglutathione pathway, the unigenes monodehydroascorbate reductase (E.C. 1.6.5.4) and glutathione reductase (E.C. 1.8.1.7) contained 2 and 3 SNPs, respectively [48]. It is likely that these mutations are associated with the high variation of vitamin C production reported between both individuals and populations of camu camu [13], as well as the differential gene expresssion and enzyme activities of the D-mannose/L-galactose pathway [120]. Our research group is currently finishing the transcriptome analysis of plantlets after germination and initial growth process and a draft genome sequence (using PacBio and Illumina technology) and annotation of camu camu. These forthcoming as well as previous functional and structural genomic resources will greatly accelerate the domestication process and the genetic improvement program of camu camu.

### Acknowledgements

We thank Dr. Jorge L. Marapara for his help with the infrastructure and equipment of Unidad Especializada de Biotecnología and Instituto Nacional de Innovación Agraria (INIA) - San Roque-Iquitos for access to the germplasm collection of camu camu. Special thanks to our students (Jhoao Flores, Jhon Vargas, Stalin Tirado, and Andry Mavila) for their great support in the design of maps, chemical structures, and metabolic pathways.

### Author details

and the genetic improvement program for camu camu. For example, using genome editing tools such as clustered regularly interspaced short palindromic repeats/associated protein-9 nuclease [CRISPR/Cas9 system], transcription activator-like effector nucleases [TALENs], and zinc finger nucleases [ZFNs] could be the molecular tools of choice to achieve the desired

To accelerate the domestication and genetic improvement program to obtain de novo elite commercial varieties, the five-step strategy of genomics-based plant germplasm research recommended by Jia et al. [119] should be implemented: (1) the detection of genomic diversity in germplasm banks, (2) the conservation and protection of germplasm based on the knowledge of genomic diversity, (3) the use of diversity information to design a representative core collection, (4) the enhancement of germplasm banks using core collections, and (5) the discov-

To date, our research team has generated fruit transcriptome data and identified several of the genes involved in vitamin C biosynthesis that have proved to be polymorphic. For example, the D-mannose/L-galactose pathway mannose-1-phosphate guanylyltransferase (E.C. 2.7.7.13)

L-galactono-1,4-lactone dehydrogenase (E.C. 1.3.2.3) only had 5 SNPs. The animal-like pathway UTP:glucose-1-phosphate uridylyltransferase (E.C. 2.7.7.9) contained 7 SNPs. In the uronic acid pathway pectin esterase (E.C. 3.1.1.11) and galacturan-1,4-alpha-galacturonidase (E.C. 3.2.1.15) showed more than 20 and 14 SNPs, respectively. Finally, in the ascorbateglutathione pathway, the unigenes monodehydroascorbate reductase (E.C. 1.6.5.4) and glutathione reductase (E.C. 1.8.1.7) contained 2 and 3 SNPs, respectively [48]. It is likely that these mutations are associated with the high variation of vitamin C production reported between both individuals and populations of camu camu [13], as well as the differential gene expresssion and enzyme activities of the D-mannose/L-galactose pathway [120]. Our research group is currently finishing the transcriptome analysis of plantlets after germination and initial growth process and a draft genome sequence (using PacBio and Illumina technology) and annotation of camu camu. These forthcoming as well as previous functional and structural genomic resources will greatly accelerate the domestication process and the genetic improve-

We thank Dr. Jorge L. Marapara for his help with the infrastructure and equipment of Unidad Especializada de Biotecnología and Instituto Nacional de Innovación Agraria (INIA) - San Roque-Iquitos for access to the germplasm collection of camu camu. Special thanks to our students (Jhoao Flores, Jhon Vargas, Stalin Tirado, and Andry Mavila) for their great support

in the design of maps, chemical structures, and metabolic pathways.


ideotypes [114–118], after obtaining the complete genome sequence of camu camu.

,50

ery of new alleles and/or genes in the core collections.

contained >20 SNPs, GDP-mannose-3<sup>0</sup>

106 Breeding and Health Benefits of Fruit and Nut Crops

ment program of camu camu.

Acknowledgements

Juan C. Castro<sup>1</sup> \*, J. Dylan Maddox2,3, Marianela Cobos4 and Sixto A. Imán<sup>5</sup>

\*Address all correspondence to: juan.castro@unapiquitos.edu.pe

1 Specialized Unit of Biotechnology, Research Center of Natural Resources of the Amazon (CIRNA), National University of the Peruvian Amazon (UNAP), Iquitos, Perú

2 Pritzker Laboratory for Molecular Systematics and Evolution, The Field Museum of Natural History, Chicago, IL, USA

3 Environmental Sciences, American Public University System, Charles Town, WV, USA

4 Laboratory of Biotechnology and Bioenergetics, Scientific University of Peru (UCP), Iquitos, Perú

5 Conservation Area of Plant Genetic Resources, National Institute of Agricultural Innovation (INIA), Iquitos, Perú

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**Chapter 6**

**Provisional chapter**

**Qualitative and Quantitative Assessment of Fatty Acids**

On the basis of gas chromatography coupled with time-of-flight mass spectrometry, we assessed the constituents and relative quantities of fatty acids extracted by supercritical carbon dioxide in seeds of hazelnut. Hazelnut seeds contain four fatty acids (palmitic, stearic, oleic, and linoleic acids). The content of unsaturated fatty acids is more than 92.9% in hazelnut seed oil. Oleic acid, which constitutes 76.1%, has a high boiling point and low volatility. Hazelnut oil has good storage stability and is recommended as senior edible oil for health and the food industry. Our study reveals the important contribution of hazelnut in the production of bioactive oils and compounds that prevent obesity, cancer, coronary disease, and many other human health as well as pharmaceutical challenges. **Keywords:** hazelnut, fatty acid, gas chromatography coupled with time-of-flight mass

**Qualitative and Quantitative Assessment of Fatty Acids** 

DOI: 10.5772/intechopen.73016

© 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 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.

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

The expanding population and improved living standards have increased the demand for edible vegetable oils. In 2016/2017, the global consumption of vegetable oils amounted to 168.53 million metric tons, compared to just 71.7 million metric tons in 1995/1996 [1]. The fastest increase has occurred in China, which in 2014 was 31.67 million metric tons, about 3.2 times greater than the consumption in 1996 [2]. Over the past decade, obesity rates, cerebrovascular, coronary disease, and cancers have increased dramatically [3–5]. This is because the changes in diets and lifestyles resulting from industrialization and market globalization have increased rapidly. However, a general improvement in the standard of living often has been

**of Hazelnut by GC-TOF/MS**

**of Hazelnut by GC-TOF/MS**

http://dx.doi.org/10.5772/intechopen.73016

Susan Mopper

**Abstract**

**1. Introduction**

Susan Mopper

Jian Ding, Chengjiang Ruan, Ying Guan and

Jian Ding, Chengjiang Ruan, Ying Guan and

Additional information is available at the end of the chapter

spectrometry, supercritical carbon dioxide extraction

Additional information is available at the end of the chapter


**Provisional chapter**

#### **Qualitative and Quantitative Assessment of Fatty Acids of Hazelnut by GC-TOF/MS of Hazelnut by GC-TOF/MS**

**Qualitative and Quantitative Assessment of Fatty Acids** 

DOI: 10.5772/intechopen.73016

Jian Ding, Chengjiang Ruan, Ying Guan and Susan Mopper Susan Mopper Additional information is available at the end of the chapter

Jian Ding, Chengjiang Ruan, Ying Guan and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73016

#### **Abstract**

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Science. 2017;256(Supplement C):120-130

116 Breeding and Health Benefits of Fruit and Nut Crops

nucleases. Trends in Plant Science. 2010;15(6):308-321

The Crop Journal. 2017;5(2):166-174

C):47-53

On the basis of gas chromatography coupled with time-of-flight mass spectrometry, we assessed the constituents and relative quantities of fatty acids extracted by supercritical carbon dioxide in seeds of hazelnut. Hazelnut seeds contain four fatty acids (palmitic, stearic, oleic, and linoleic acids). The content of unsaturated fatty acids is more than 92.9% in hazelnut seed oil. Oleic acid, which constitutes 76.1%, has a high boiling point and low volatility. Hazelnut oil has good storage stability and is recommended as senior edible oil for health and the food industry. Our study reveals the important contribution of hazelnut in the production of bioactive oils and compounds that prevent obesity, cancer, coronary disease, and many other human health as well as pharmaceutical challenges.

**Keywords:** hazelnut, fatty acid, gas chromatography coupled with time-of-flight mass spectrometry, supercritical carbon dioxide extraction

#### **1. Introduction**

The expanding population and improved living standards have increased the demand for edible vegetable oils. In 2016/2017, the global consumption of vegetable oils amounted to 168.53 million metric tons, compared to just 71.7 million metric tons in 1995/1996 [1]. The fastest increase has occurred in China, which in 2014 was 31.67 million metric tons, about 3.2 times greater than the consumption in 1996 [2]. Over the past decade, obesity rates, cerebrovascular, coronary disease, and cancers have increased dramatically [3–5]. This is because the changes in diets and lifestyles resulting from industrialization and market globalization have increased rapidly. However, a general improvement in the standard of living often has been

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

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

accompanied by unhealthy dietary patterns and insufficient physical activity to maintain an optimal energy balance and a healthy weight. The net result has been increased prevalence of diet-related chronic diseases.

**2.2. Materials**

We collected seeds of hazelnut hybrids (*Corylus heterophylla* × *C. avellana*) from Liaoning Provinces of China (**Figure 1A**). Oils from seeds were extracted by SCDE (**Figure 1C**). Seeds were stored in closed plastic bags in dark at 4°C. Commercially prepared seed oils were pro-

Seed samples were powdered using laboratory plant grinder and air-dried [16]. Ground sample (100 g) was then placed into the extraction kettle of SFT-110 supercritical carbon dioxide extracting device from Supercritical Fluid Technologies, Inc. (Newark, USA). The pressure parameter and temperature of kettle heating were set at 5500 PSI and 60°C, respectively. The

Fatty acid profile was determined as fatty acid methyl esters (FAME) by gas chromatography. The methyl esters were prepared according to Wang et al. [18] and Sanchez-Salcedo et al. [6] with some modifications. Twenty milligram of oil was added to a test tube, followed by the addition of 2 mL of n-hexane and 5 mL of methanol-potassium hydroxide solution (1 M). The mixture was placed in a blender shock with water bath at 60°C for 30 min. After the reaction,

left at 60°C for 30 min. FAME were then extracted using saturated sodium chloride solution (2 mL) and n-hexane (2 mL) through vigorous shaking for 1 min. Top layer was transferred

The fatty acid compositions were analyzed using a Clarus 680 GC coupled with AxION iQT TOF/MS system (PerkinElmer, Shelton, USA). The system was equipped with Agilent J&W DB-23 capillary column (60 m × 0.25 mm × 0.25 μm). The flow rate of carrier gas (Helium) was 1 mL·min−1 with a split mode (1:20). The temperature program was started at 50°C, raised

**Figure 1.** Fruits (A), fatty acid composition (B), and oils (C) of hazelnut. HE, oils extracted from seeds of hazelnut; HP,

) in methanol was added to the mixture, and the samples were

Qualitative and Quantitative Assessment of Fatty Acids of Hazelnut by GC-TOF/MS

http://dx.doi.org/10.5772/intechopen.73016

119

vided by Dihao (Liaoyang, China) company (**Figure 1C**).

carbon dioxide flow and extraction time followed was 18 mL·min−1 [17].

**2.3. Oil extraction by supercritical carbon dioxide**

**2.4. Determination of fatty acids**

10 mL of boron trifluoride (BF3

into a vial and stored at −20°C.

commercially prepared product oil of hazelnut seed.

Fatty acids form the building blocks of lipid molecules, contribute to the structure of cell membranes and hormones, and provide cells with energy [6]. Palmitic acid (C16:0) and stearic acid (C18:0) are common saturated fatty acids (SFA) in edible oils and are thought to raise blood cholesterol and low-density lipoprotein (LDL) levels, leading to many diseases [7]. Monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) are considered a healthy source of dietary fat for humans. Originally, the American Heart Association (AHA) recommended a fatty acid balance of approximately 1:1.5:1 ratio of SFA:MUFA:PUFA [8]. Oleic acid (an omega-9 MUFA) is defined as "conditionally essential," because it can be synthesized *in vivo*. Any amount of omega-9 is beneficial [9]. Linoleic acid (an omega-6 PUFA) is a desirable and an essential fatty acid, as humans cannot synthesize double bonds in the n-6 positions of their hydrocarbon chains.

Hazelnut (*Corylus avellana*) is the second most popular nut worldwide, and it is distributed in several areas of Europe and Asia [10–14]. Hazelnut seeds are high energy food rich in fats as well as proteins. Seed oil contents range from 41.96 to 63.73% of hazelnut kernel dry weight [11], and fatty acids of hazelnut are similar in composition to those of olive oil. The nutrition and health benefits of UFA in hazelnut oils can reduce or prevent cancer, cardiovascular, and autoimmune diseases, and have anti-ulcerogenic, regenerating, and anti-inflammatory properties [3, 10–13]. Hazelnut seeds contain high concentrations of bioactive compounds (such as tocopherols, polyphenolics, neolignans, and 1,1-diphenyl-2-picrylhydrazyl radical) [10–14]. These are valuable sources of phytonutrients, fiber, and antioxidants [15].

Few studies have compared the fatty acid composition of seed oils extracted by supercritical carbon dioxide extracting method (SCDE) and commercially prepared product oils. In this chapter, we (1) extracted seed oils using SCDE, (2) identified the constituents and relative contents of fatty acids in the extracted oils and in products purchased from Dihao company in China, using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF/ MS), and (3) compared the differences in fatty acids between extracted oils and commercially prepared product oils. Finally, we also discuss the beneficial functions of these oils and provide useful information for producing this bioactive oil that reduces or prevents obesity, cancer, coronary disease, and many other human health as well as pharmaceutical challenges.

### **2. Materials and methods**

#### **2.1. Chemicals and reagents**

Carbon dioxide gas (99.999%) was purchased from Airichen (Dalian, China). Hexane, methanol, and methylene chloride (≥97%, GC grade) were obtained from Honeywell (Ulsan, Korea). The boron trifluoride and fatty acid methyl ester mix was obtained from Sigma-Aldrich (Steinheim, Germany).

#### **2.2. Materials**

accompanied by unhealthy dietary patterns and insufficient physical activity to maintain an optimal energy balance and a healthy weight. The net result has been increased prevalence of

Fatty acids form the building blocks of lipid molecules, contribute to the structure of cell membranes and hormones, and provide cells with energy [6]. Palmitic acid (C16:0) and stearic acid (C18:0) are common saturated fatty acids (SFA) in edible oils and are thought to raise blood cholesterol and low-density lipoprotein (LDL) levels, leading to many diseases [7]. Monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) are considered a healthy source of dietary fat for humans. Originally, the American Heart Association (AHA) recommended a fatty acid balance of approximately 1:1.5:1 ratio of SFA:MUFA:PUFA [8]. Oleic acid (an omega-9 MUFA) is defined as "conditionally essential," because it can be synthesized *in vivo*. Any amount of omega-9 is beneficial [9]. Linoleic acid (an omega-6 PUFA) is a desirable and an essential fatty acid, as humans cannot synthesize double bonds in the n-6

Hazelnut (*Corylus avellana*) is the second most popular nut worldwide, and it is distributed in several areas of Europe and Asia [10–14]. Hazelnut seeds are high energy food rich in fats as well as proteins. Seed oil contents range from 41.96 to 63.73% of hazelnut kernel dry weight [11], and fatty acids of hazelnut are similar in composition to those of olive oil. The nutrition and health benefits of UFA in hazelnut oils can reduce or prevent cancer, cardiovascular, and autoimmune diseases, and have anti-ulcerogenic, regenerating, and anti-inflammatory properties [3, 10–13]. Hazelnut seeds contain high concentrations of bioactive compounds (such as tocopherols, polyphenolics, neolignans, and 1,1-diphenyl-2-picrylhydrazyl radical) [10–14].

Few studies have compared the fatty acid composition of seed oils extracted by supercritical carbon dioxide extracting method (SCDE) and commercially prepared product oils. In this chapter, we (1) extracted seed oils using SCDE, (2) identified the constituents and relative contents of fatty acids in the extracted oils and in products purchased from Dihao company in China, using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF/ MS), and (3) compared the differences in fatty acids between extracted oils and commercially prepared product oils. Finally, we also discuss the beneficial functions of these oils and provide useful information for producing this bioactive oil that reduces or prevents obesity, cancer, coronary disease, and many other human health as well as pharmaceutical challenges.

Carbon dioxide gas (99.999%) was purchased from Airichen (Dalian, China). Hexane, methanol, and methylene chloride (≥97%, GC grade) were obtained from Honeywell (Ulsan, Korea). The boron trifluoride and fatty acid methyl ester mix was obtained from Sigma-Aldrich

These are valuable sources of phytonutrients, fiber, and antioxidants [15].

diet-related chronic diseases.

118 Breeding and Health Benefits of Fruit and Nut Crops

positions of their hydrocarbon chains.

**2. Materials and methods**

**2.1. Chemicals and reagents**

(Steinheim, Germany).

We collected seeds of hazelnut hybrids (*Corylus heterophylla* × *C. avellana*) from Liaoning Provinces of China (**Figure 1A**). Oils from seeds were extracted by SCDE (**Figure 1C**). Seeds were stored in closed plastic bags in dark at 4°C. Commercially prepared seed oils were provided by Dihao (Liaoyang, China) company (**Figure 1C**).

#### **2.3. Oil extraction by supercritical carbon dioxide**

Seed samples were powdered using laboratory plant grinder and air-dried [16]. Ground sample (100 g) was then placed into the extraction kettle of SFT-110 supercritical carbon dioxide extracting device from Supercritical Fluid Technologies, Inc. (Newark, USA). The pressure parameter and temperature of kettle heating were set at 5500 PSI and 60°C, respectively. The carbon dioxide flow and extraction time followed was 18 mL·min−1 [17].

#### **2.4. Determination of fatty acids**

Fatty acid profile was determined as fatty acid methyl esters (FAME) by gas chromatography. The methyl esters were prepared according to Wang et al. [18] and Sanchez-Salcedo et al. [6] with some modifications. Twenty milligram of oil was added to a test tube, followed by the addition of 2 mL of n-hexane and 5 mL of methanol-potassium hydroxide solution (1 M). The mixture was placed in a blender shock with water bath at 60°C for 30 min. After the reaction, 10 mL of boron trifluoride (BF3 ) in methanol was added to the mixture, and the samples were left at 60°C for 30 min. FAME were then extracted using saturated sodium chloride solution (2 mL) and n-hexane (2 mL) through vigorous shaking for 1 min. Top layer was transferred into a vial and stored at −20°C.

The fatty acid compositions were analyzed using a Clarus 680 GC coupled with AxION iQT TOF/MS system (PerkinElmer, Shelton, USA). The system was equipped with Agilent J&W DB-23 capillary column (60 m × 0.25 mm × 0.25 μm). The flow rate of carrier gas (Helium) was 1 mL·min−1 with a split mode (1:20). The temperature program was started at 50°C, raised

**Figure 1.** Fruits (A), fatty acid composition (B), and oils (C) of hazelnut. HE, oils extracted from seeds of hazelnut; HP, commercially prepared product oil of hazelnut seed.

to 200°C at 15°C·min−1 up, and finally to 230°C for 10 min. The temperature of EI ion source was 230°C, and the injection volume was 1 μL. Fatty acids were identified based on the mass spectra of 37 FAME standards. Fatty acid composition was expressed using peak area normalization method. All the analyses were conducted in three replicates.

Koksal et al. [20] investigated the oleic acid contents (74.2–82.8%) among 17 different hazelnut varieties grown in the Black Sea Region of Turkey. Because of its high percentage of oleic acid, hazelnut oil is stable edible oil and considered beneficial for a healthy diet. Oleic acid naturally exists in many plant and animal products and is considered one of the healthiest sources of dietary fat. It can reduce ratios of LDL/HDL and triglycerides in the blood, prevent coronary heart disease, hypertension, cerebrovascular disease, arteriosclerosis, stomach ache, and burn injuries; a high MUFA diet is recommended in diabetes mellitus patients [24]. PUFA are precursors of potent lipid mediators, important structural components of cell membranes, and play an important role in inflammation regulation and cell function [25]. Omega-6 fatty acids are necessary for healthy brain function, skin and hair growth, bone density, energy production, and reproductive health. Meat, eggs, and nut-based oils are the main dietary sources of omega-6 fatty acids [26]. The linoleic acid (one of omega-6 fatty acids) concentrations in HE and HP oils was 16.8 and 19.7%, respectively (**Table 1**). The linoleic acid concentration of HP oil was significantly higher than HE oil. Bacchetta et al. [11] found the percentage of linoleic acid ranged from 5.91 to 19.01% among 75 European hazelnut germplasm oil samples and detected its content was inversely correlated with oleic acid, because oleic acid is the precursor of linoleic and linolenic acids [11]. Because of the high level of oleic and linoleic acids, the composition of TUFA was more than 92%

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**Fatty acids HE HP Dif. Std. Student** *t* Palmitic acid (C16:0) 4.73 ± 0.03 3.33 ± 0.22 1.40 0.16 0.71\*\* Stearic acid (C18:0) 2.40 ± 0.08 2.39 ± 0.14 0.01 0.11 0.07 Oleic acid (C18:1) 76.07 ± 0.79 74.54 ± 0.62 1.53 0.71 2.66 Linoleic acid (C18:2) 16.80 ± 0.84 19.74 ± 0.98 −2.94 0.91 3.96\* SFA 7.13 ± 0.05 5.72 ± 0.36 1.41 0.26 6.72\* MUFA 76.07 ± 0.79 74.54 ± 0.62 1.53 0.71 2.66 PUFA 16.80 ± 0.84 19.74 ± 0.98 −2.94 0.91 3.96\* TUFA 92.87 ± 0.05 94.28 ± 0.36 −1.41 0.26 6.72\* MUFA/SFA 10.67 ± 0.04 13.03 ± 0.72 −2.39 0.51 5.79\* PUFA/SFA 2.35 ± 0.14 3.45 ± 0.39 −1.11 0.29 4.72\*\*

Data expressed as mean ± standard error (n = 3); nd, not detected; HE, oils extracted from seeds of hazelnut; HP, commercially prepared product oil of hazelnut seed; SFA, saturated fatty acids, are the sum of palmitic and stearic acid; MUFA, monounsaturated fatty acids, are the sum of stearic, eicosenoic, erucic, and nervonic acids; PUFA, polyunsaturated fatty acids, are the sum of linoleic and linolenic acids; MUFA/SFA, monounsaturated/saturated fatty acids ratio; PUFA/SFA, polyunsaturated/saturated fatty acids ratio; Dif., difference between oils extracted from seeds

**Table 1.** Fatty acid composition (weight % of total fatty acids) and comparison of the mean values (%) of fatty acids

and commercially prepared product oils; Std, standard deviation (n = 3)

between the oils extracted from seeds and the commercially prepared product oils.

\* *P* < 0.05. \*\**P* < 0.01.

#### **2.5. Statistical analysis**

The results were expressed as mean ± standard deviation (*n* = 3). The *p*-value ≤0.05 was used to denote significant differences between mean values determined by one-way analysis of variance (ANOVA). All statistical analyses were performed using SPSS Statistics 20.0 software (IBM SPSS Statistics 20.0, Armonk, NY, USA) [19].

### **3. Results and discussion**

#### **3.1. Hazelnut species and distribution in China**

*C. America*, *C. avellana*, *C. colurna*, and *C. mandshurica* are widely distributed species in the world. The world's hazelnut production is mainly covered by two main market players (Turkey and Italy). Turkey is the major hazelnut (*C. avellana*) producing country, supplying 65% of the world's total production. However, USA, Azerbaijan, Georgia, China, Iran, Spain, France, Kirgizstan, Poland, and Croatia are smaller but significant producers [15, 20]. *C. mandshurica*, also known as pilose hazelnut, is an economically and ecologically important species in China [21]. There is currently more than 4 million acres of natural hazel groves in northeastern China alone. Zong et al. [21] applied 10 polymorphic simple sequence repeat (SSR) markers to evaluate the genetic diversity and population structure of 348 *C. mandshurica* individuals among 12 populations in China and found that there was obvious genetic differentiation among populations from Northeast China to North China. The hybrid varieties of *C. heterophylla* and *C. avellana* have been widely planted in North China because of the cold resistance and high yield, which are superior to *C. avellana* in terms of unsaturated fatty acid content and antioxidant activity [22, 23].

#### **3.2. Fatty acid composition in hazelnut seed oils**

Hazelnuts are a high energy food with functional fats and proteins, which are the main components of the hazelnut kernel. The lipid portion represents a major determinant of kernel flavor, particularly following roasting [11]. We determined four fatty acids in hazelnut seed oils by GC-TOF/MS (**Figure 1B**). Oleic acid was the main fatty acid followed by linoleic acid, and palmitic and stearic acids were also measured in hazelnut oil. Hazelnut oil has low concentrations of SFA, and palmitic and stearic acids (4.7 and 2.4%, respectively) were quantified in hazelnut oil. Palmitic acid and SFA concentrations of oils extracted by the method of SCDE were significantly higher than in the commercially prepared product (**Table 1**).

Oleic acid (C18:1) concentrations were 76.1 and 74.5% in oils extracted from seeds of hazelnut (HE) and commercially prepared product oil of hazelnut seed (HP, **Table 1**), respectively. Koksal et al. [20] investigated the oleic acid contents (74.2–82.8%) among 17 different hazelnut varieties grown in the Black Sea Region of Turkey. Because of its high percentage of oleic acid, hazelnut oil is stable edible oil and considered beneficial for a healthy diet. Oleic acid naturally exists in many plant and animal products and is considered one of the healthiest sources of dietary fat. It can reduce ratios of LDL/HDL and triglycerides in the blood, prevent coronary heart disease, hypertension, cerebrovascular disease, arteriosclerosis, stomach ache, and burn injuries; a high MUFA diet is recommended in diabetes mellitus patients [24].

PUFA are precursors of potent lipid mediators, important structural components of cell membranes, and play an important role in inflammation regulation and cell function [25]. Omega-6 fatty acids are necessary for healthy brain function, skin and hair growth, bone density, energy production, and reproductive health. Meat, eggs, and nut-based oils are the main dietary sources of omega-6 fatty acids [26]. The linoleic acid (one of omega-6 fatty acids) concentrations in HE and HP oils was 16.8 and 19.7%, respectively (**Table 1**). The linoleic acid concentration of HP oil was significantly higher than HE oil. Bacchetta et al. [11] found the percentage of linoleic acid ranged from 5.91 to 19.01% among 75 European hazelnut germplasm oil samples and detected its content was inversely correlated with oleic acid, because oleic acid is the precursor of linoleic and linolenic acids [11]. Because of the high level of oleic and linoleic acids, the composition of TUFA was more than 92%


Data expressed as mean ± standard error (n = 3); nd, not detected; HE, oils extracted from seeds of hazelnut; HP, commercially prepared product oil of hazelnut seed; SFA, saturated fatty acids, are the sum of palmitic and stearic acid; MUFA, monounsaturated fatty acids, are the sum of stearic, eicosenoic, erucic, and nervonic acids; PUFA, polyunsaturated fatty acids, are the sum of linoleic and linolenic acids; MUFA/SFA, monounsaturated/saturated fatty acids ratio; PUFA/SFA, polyunsaturated/saturated fatty acids ratio; Dif., difference between oils extracted from seeds and commercially prepared product oils; Std, standard deviation (n = 3) \*

to 200°C at 15°C·min−1 up, and finally to 230°C for 10 min. The temperature of EI ion source was 230°C, and the injection volume was 1 μL. Fatty acids were identified based on the mass spectra of 37 FAME standards. Fatty acid composition was expressed using peak area normal-

The results were expressed as mean ± standard deviation (*n* = 3). The *p*-value ≤0.05 was used to denote significant differences between mean values determined by one-way analysis of variance (ANOVA). All statistical analyses were performed using SPSS Statistics 20.0 software

*C. America*, *C. avellana*, *C. colurna*, and *C. mandshurica* are widely distributed species in the world. The world's hazelnut production is mainly covered by two main market players (Turkey and Italy). Turkey is the major hazelnut (*C. avellana*) producing country, supplying 65% of the world's total production. However, USA, Azerbaijan, Georgia, China, Iran, Spain, France, Kirgizstan, Poland, and Croatia are smaller but significant producers [15, 20]. *C. mandshurica*, also known as pilose hazelnut, is an economically and ecologically important species in China [21]. There is currently more than 4 million acres of natural hazel groves in northeastern China alone. Zong et al. [21] applied 10 polymorphic simple sequence repeat (SSR) markers to evaluate the genetic diversity and population structure of 348 *C. mandshurica* individuals among 12 populations in China and found that there was obvious genetic differentiation among populations from Northeast China to North China. The hybrid varieties of *C. heterophylla* and *C. avellana* have been widely planted in North China because of the cold resistance and high yield, which are superior to *C. avellana* in terms of unsaturated fatty acid

Hazelnuts are a high energy food with functional fats and proteins, which are the main components of the hazelnut kernel. The lipid portion represents a major determinant of kernel flavor, particularly following roasting [11]. We determined four fatty acids in hazelnut seed oils by GC-TOF/MS (**Figure 1B**). Oleic acid was the main fatty acid followed by linoleic acid, and palmitic and stearic acids were also measured in hazelnut oil. Hazelnut oil has low concentrations of SFA, and palmitic and stearic acids (4.7 and 2.4%, respectively) were quantified in hazelnut oil. Palmitic acid and SFA concentrations of oils extracted by the method of SCDE

Oleic acid (C18:1) concentrations were 76.1 and 74.5% in oils extracted from seeds of hazelnut (HE) and commercially prepared product oil of hazelnut seed (HP, **Table 1**), respectively.

were significantly higher than in the commercially prepared product (**Table 1**).

ization method. All the analyses were conducted in three replicates.

(IBM SPSS Statistics 20.0, Armonk, NY, USA) [19].

**3.1. Hazelnut species and distribution in China**

content and antioxidant activity [22, 23].

**3.2. Fatty acid composition in hazelnut seed oils**

**2.5. Statistical analysis**

120 Breeding and Health Benefits of Fruit and Nut Crops

**3. Results and discussion**

\*\**P* < 0.01.

**Table 1.** Fatty acid composition (weight % of total fatty acids) and comparison of the mean values (%) of fatty acids between the oils extracted from seeds and the commercially prepared product oils.

*P* < 0.05.

(**Table 1**). Ciemniewska-Zytkiewicz et al. [15] quantified the concentrations of TUFA and oleic acid as 94.01 and 80.25%, respectively, in hazelnut seed oil, which were higher than the previous reports.

**Author details**

, Chengjiang Ruan1

sumption [Accessed: Sep 1, 2017]

DOI: 10.1016/j.metabol.2008.04.012

2014;**160**:398-406. DOI: 10.7326/M13-1788

s13596-011-0002-x

\*Address all correspondence to: ruan@dlnu.edu.cn

\*, Ying Guan2

3 Department of Biology, University of Louisiana, Lafayette, USA

2006;**60**:502-507. DOI: 10.1016/j.biopha.2006.07.080

1 Institute of Plant Resources, Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian Minzu University, Dalian, P.R. China

2 Institute of Berries, Heilongjiang Academy of Agricultural Sciences, Suiling, P.R. China

[1] Statista. Vegetable Oils: Global Consumption by Oil Type 1995-2017 [Internet]. 2017. Available from: http://www.statista.com/statistics/263937/vegetable-oils-global-con-

[2] Wang RY. Review on the production, consumption and development of grain, oil and oil

[3] Simopoulos AP.Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: Nutritional implications for chronic diseases. Biomedicine & Pharmacotherapy.

[4] Jones PJH, Jew S, Abu-Mweis S. The effect of dietary oleic, linoleic, and linolenic acids on fat oxidation and energy expenditure in healthy men. Metabolism. 2008;**57**:1198-1203.

[5] Chowdhury R, Warnakula S, Kunutsor S, Crowe F, Ward HA, Johnson L, Franco OH, Butterworth AS, Forouhi NG, Thompson SG, Khaw KT, Mozaffarian D, Danesh J, Di Angelantonio E. Association of dietary, circulating, and supplement fatty acids with coronary risk: A systematic review and meta-analysis. Annals of Internal Medicine.

[6] Sanchez-Salcedo EM, Sendra E, Carbonell-Barrachina AA, Martinez JJ, Hernandez F. Fatty acids composition of Spanish black (*Morus nigra* L.) and white (*Morus alba* L.) mul-

[7] Hayes KC. Dietary fat and heart health: In search of the ideal fat. Asia Pacific. The Journal

[8] Grundy SM. What is the desirable ratio of saturated, polyunsaturated, and monounsaturated fatty acids in the diet? The American Journal of Clinical Nutrition. 1997;**66**:988-990

[9] Asif M. Health effects of omega-3, 6, 9 fatty acids: *Perilla frutescens* is a good example of plant oils. Oriental Pharmacy and Experimental Medicine. 2011;**11**:51-59. DOI: 10.1007/

berries. Food Chemistry. 2016;**190**:566-571. DOI: 10.1016/j.foodchem.2015.06.008

of Clinical Nutrition. 2002;**11**:394-400. DOI: 10.1371/journal.pone.0096186

industry of China in 2015. Cereals Oils Proc. 2015;**5**:14-19 (In Chinese)

and Susan Mopper3

Qualitative and Quantitative Assessment of Fatty Acids of Hazelnut by GC-TOF/MS

http://dx.doi.org/10.5772/intechopen.73016

123

Jian Ding1

**References**

#### **3.3. Fatty acid ratios in hazelnut seed oils**

Fatty acids and their ratios affect lipid oxidation and physiological functions of oils and fats [7, 8]. Hazelnut oils have low concentrations of SFA and high concentrations of MUFA. The main sources of SFA in food are animal products (such as milk, meat, salmon, and egg yolks) and some plant products (such as chocolate and cocoa butter, coconut, and palm kernel oils). In modern time, people can easily consume sufficient SFA. SFA are thought to raise total cholesterol (TC) and low-density lipoprotein (LDL), which are undesirable to human health. But certain SFA (as consumed in our daily diet) have beneficial effects on the ratio of LDL to high-density lipoprotein (HDL) [7]. Recently, Souza et al. [27] and Mancini et al. [28] showed that saturated fat intake was not associated with mortality, cardiovascular disease, coronary heart disease (CHD), ischemic stroke, or type 2 diabetes, whereas trans fats were associated with mortality, total CHD, and CHD mortality, probably because of higher levels of intake of industrial trans fats than ruminant trans fats. No trans fatty acid occurred in hazelnut oil, and it had high MUFA/SFA (10.67) and low PUFA/SFA ratio (2.35) in HE oil (**Table 1**). So hazelnut oil has a high boiling point and low volatility and can improve the nutritional quality and shelf-life of processed foods [11]. Hazelnut oil has good storage stability and is recommended as senior edible oil for health and the food industry.

In addition, the abundant tocopherol, sterol, phenolic compounds, fiber, and antioxidants such as Vitamin E were also determined [29]. Hazelnut oil is suitable for cooking, salad oils, and for the manufacture of margarine. Meanwhile, a high level of MUFA and a low quantity of SFA in hazelnut oil enhance its usefulness in food as well as oleochemical applications [29].

#### **4. Conclusions**

Hazelnut seed oil contains four fatty acids, and the content of unsaturated fatty acid is more than 92.9% in hazelnut seed oil. The oleic acid concentration is 76.1%, which has a high boiling point and low volatility. Hazelnut oil is recommended as senior edible oil for health and the food industry. Our study reveals the important contribution of hazelnut for producing bioactive oils and compounds that reduce or prevent obesity, cancer, coronary disease, and many other human health as well as pharmaceutical challenges.

### **Acknowledgements**

This research was funded by the National Natural Science Foundation of China (Project No. 31570681) and a Marie Curie International Incoming Fellowship from the 7th European Community Framework Program (Project Nos. PIIF-GA-2010-272048 and PIIFR-GA-2010-910048).

### **Author details**

(**Table 1**). Ciemniewska-Zytkiewicz et al. [15] quantified the concentrations of TUFA and oleic acid as 94.01 and 80.25%, respectively, in hazelnut seed oil, which were higher than

Fatty acids and their ratios affect lipid oxidation and physiological functions of oils and fats [7, 8]. Hazelnut oils have low concentrations of SFA and high concentrations of MUFA. The main sources of SFA in food are animal products (such as milk, meat, salmon, and egg yolks) and some plant products (such as chocolate and cocoa butter, coconut, and palm kernel oils). In modern time, people can easily consume sufficient SFA. SFA are thought to raise total cholesterol (TC) and low-density lipoprotein (LDL), which are undesirable to human health. But certain SFA (as consumed in our daily diet) have beneficial effects on the ratio of LDL to high-density lipoprotein (HDL) [7]. Recently, Souza et al. [27] and Mancini et al. [28] showed that saturated fat intake was not associated with mortality, cardiovascular disease, coronary heart disease (CHD), ischemic stroke, or type 2 diabetes, whereas trans fats were associated with mortality, total CHD, and CHD mortality, probably because of higher levels of intake of industrial trans fats than ruminant trans fats. No trans fatty acid occurred in hazelnut oil, and it had high MUFA/SFA (10.67) and low PUFA/SFA ratio (2.35) in HE oil (**Table 1**). So hazelnut oil has a high boiling point and low volatility and can improve the nutritional quality and shelf-life of processed foods [11]. Hazelnut oil has good storage stability and is recommended

In addition, the abundant tocopherol, sterol, phenolic compounds, fiber, and antioxidants such as Vitamin E were also determined [29]. Hazelnut oil is suitable for cooking, salad oils, and for the manufacture of margarine. Meanwhile, a high level of MUFA and a low quantity of SFA in hazelnut oil enhance its usefulness in food as well as oleochemical appli-

Hazelnut seed oil contains four fatty acids, and the content of unsaturated fatty acid is more than 92.9% in hazelnut seed oil. The oleic acid concentration is 76.1%, which has a high boiling point and low volatility. Hazelnut oil is recommended as senior edible oil for health and the food industry. Our study reveals the important contribution of hazelnut for producing bioactive oils and compounds that reduce or prevent obesity, cancer, coronary disease, and many

This research was funded by the National Natural Science Foundation of China (Project No. 31570681) and a Marie Curie International Incoming Fellowship from the 7th European Community

Framework Program (Project Nos. PIIF-GA-2010-272048 and PIIFR-GA-2010-910048).

the previous reports.

cations [29].

**4. Conclusions**

**Acknowledgements**

**3.3. Fatty acid ratios in hazelnut seed oils**

122 Breeding and Health Benefits of Fruit and Nut Crops

as senior edible oil for health and the food industry.

other human health as well as pharmaceutical challenges.

Jian Ding1 , Chengjiang Ruan1 \*, Ying Guan2 and Susan Mopper3

\*Address all correspondence to: ruan@dlnu.edu.cn

1 Institute of Plant Resources, Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian Minzu University, Dalian, P.R. China

2 Institute of Berries, Heilongjiang Academy of Agricultural Sciences, Suiling, P.R. China

3 Department of Biology, University of Louisiana, Lafayette, USA

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extraction and pre-

Food Chemistry. 2008;**106**:896-904. DOI: 10.1016/j.foodchem.2007.06.058

[17] Chen J, Li J, Sun AD, Zhang BL, Qin SG, Zhang YQ. Supercritical CO2

ADILAN. Trials. 2014;**15**:2. DOI: 10.1186/1745-6215-15-2

2016;**53**:3522-3531. DOI: 10.1007/s13197-016-2329-2

2013;**191**:57-73. DOI: 10.1007/s10681-013-0861-y

Sciences. 2017;**18**:392. DOI: 10.3390/ijms18020392

**57**:4645-4650. DOI: 10.1021/jf900489d

DOI: 10.1010/j.jfca.2015.08.010

124 Breeding and Health Benefits of Fruit and Nut Crops

indcrop.2014.06.013

indcrop.2015.08.021

foodchem.2005.08.013


**Chapter 7**

**Provisional chapter**

**Clinical Applications of Pomegranate**

**Clinical Applications of Pomegranate**

DOI: 10.5772/intechopen.75962

Pomegranate, *Punica granatum* L., is an ancient, unique fruit borne on a small, long-living tree in the Mediterranean region, Southeast Asia, and tropical Africa. Pomegranate was mentioned in ancient times in the Old Bible, the Jewish Torah, and mentioned three times in the holy Quran where it was described as one of the paradise fruits. In ayurvedic medicine, pomegranate is used in the treatment of parasitic infection, diarrhea, and ulcers. Recently, pomegranate has been studied in several systems of medicine for its pharmacological actions: anti-inflammatory, antioxidant, and anticarcinogenic. The aim of the chapter is to summarize pomegranate efficacy in many preclinical and

**Keywords:** pomegranate, ayurvedic medicine, pharmacological activities, preclinical,

Pomegranate*,* (*Punica granatum* L.), a paradise fruit, has a great value throughout history. It had been mentioned in Judaism, Christianity, and Islamic religions [1]. From ancient times, pomegranate was used in treatment of diarrhea [2], parasitic infections [3], and diabetes mellitus [4]. Greco-Arab and Islamic medicine prescribed pomegranate for sore throat, inflammation, and rheumatism [5]. Various pomegranate activities (anti-inflammatory, antioxidant, and anticancer) encouraged growing number of studies to apply it in solving multiple medical problems [6]. Pomegranate plant is a small tree (**Figure 1**) that is cultivated in the Middle East, Mediterranean region, China, India, California, and Mexico. The fruit (**Figure 2**) is com-

posed of many parts such as seeds, peels (pericarp), pulp, and juice [6].

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

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

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

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.75962

Sally Elnawasany

Sally Elnawasany

**Abstract**

clinical studies.

clinical studies

**1. Introduction**

#### **Clinical Applications of Pomegranate Clinical Applications of Pomegranate**

#### Sally Elnawasany Sally Elnawasany

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.75962

**Abstract**

Pomegranate, *Punica granatum* L., is an ancient, unique fruit borne on a small, long-living tree in the Mediterranean region, Southeast Asia, and tropical Africa. Pomegranate was mentioned in ancient times in the Old Bible, the Jewish Torah, and mentioned three times in the holy Quran where it was described as one of the paradise fruits. In ayurvedic medicine, pomegranate is used in the treatment of parasitic infection, diarrhea, and ulcers. Recently, pomegranate has been studied in several systems of medicine for its pharmacological actions: anti-inflammatory, antioxidant, and anticarcinogenic. The aim of the chapter is to summarize pomegranate efficacy in many preclinical and clinical studies.

DOI: 10.5772/intechopen.75962

**Keywords:** pomegranate, ayurvedic medicine, pharmacological activities, preclinical, clinical studies

#### **1. Introduction**

Pomegranate*,* (*Punica granatum* L.), a paradise fruit, has a great value throughout history. It had been mentioned in Judaism, Christianity, and Islamic religions [1]. From ancient times, pomegranate was used in treatment of diarrhea [2], parasitic infections [3], and diabetes mellitus [4]. Greco-Arab and Islamic medicine prescribed pomegranate for sore throat, inflammation, and rheumatism [5]. Various pomegranate activities (anti-inflammatory, antioxidant, and anticancer) encouraged growing number of studies to apply it in solving multiple medical problems [6]. Pomegranate plant is a small tree (**Figure 1**) that is cultivated in the Middle East, Mediterranean region, China, India, California, and Mexico. The fruit (**Figure 2**) is composed of many parts such as seeds, peels (pericarp), pulp, and juice [6].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*2.1.1. Gastrointestinal inflammation*

*Helicobacter pylori* (*H. pylori)* are major etiological agents in peptic ulcer. Pomegranate methanol extract produced a remarkable anti-*H. pylori* activity with mean diameter of inhibition zone 39 at 100 μg disc−1 [8]. This activity is explained by altering bacterial cell surface hydrophobicity and prevention of bacterial adhesion to gastric mucosa [9]. Moreover, pomegranate revealed gastroprotective potential via antioxidant mechanism in aspirin- and ethanol-induced gastric ulceration in animal models [10]. Gastroprotective property of pomegranate is attributed to its constituents (saponin, tannins, and flavonoids) as demonstrated in another study on wistar rats where oral administration (490 and 980 mg/kg body weight) of pomegranate aqueous methanolic extract significantly reduced gastric ulcer index in alcohol-, indomethacin-, and aspirininduced ulcers [11]. Tannins are high molecular weight phenolic compounds present in many plants, including pomegranate fruit pericarp (peels). These compounds have the capacity to form complexes mainly with proteins [12, 13]. Pomegranate tannins form a protective layer (tannin-protein/tannin-polysaccharide complex), upon damaged epithelial tissues, thus allowing the healing process below to occur naturally through prevention of bleeding and acceleration of ulcer healing [14, 15]. All parts of the pomegranate tree have been used as a source for tannin in the leather industry, changing animal hide into leather. About 10–25% of tannin are present in the trunk bark and were important in leather production in Morocco. In this process, collagen chains in the hide are cross-linked by tannin to give leather. The formation of various complex bonds helps the tannin-protein polymer combination [16, 17]. These facts take our attention to the Islamic advice "To *eat pomegranate with its pericarp as it is tanning for the stomach*".

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 129

Inflammatory response is induced by transduction cascades initiated by many inflammatory mediators, that is, tumor necrosis factor α (TNF-α) and nuclear factor κB (NF-κB). Pomegranate inhibited TNF-α-induced NF-κB activation and COX-2 expression in colon cell line. This effect was highly presented by pomegranate juice compared to single constituents, that is, tannin and punicalagin. This highlights the synergism between all bioactive pomegranate compounds [18]. Prebiotics are food agents that stimulate the growth or activity of beneficial microorganisms. Pomegranate peel extract (6 mg/d for 4 weeks) increased the cecal pool of beneficial bifidobacteria when given to high-fat diet mice. Additionally, it counteracted the high-fat-induced expression of inflammatory markers both in the colon and in the visceral adipose tissue [19]. Through its antioxidative action, pomegranate elagic acid (EA) (10 mg/kg) in colonic-delivering microsphere significantly ameliorated the severity of colonic lesions and reduced myeloperoxidase (MPO) activity and lipid peroxidation. This effect was obtained by orally administrating it to rat model of dextran sulfate sodium (DSS)-induced ulcerative colitis [20]. Mast cells are

*2.1.1.1. Gastric inflammation*

*2.1.1.1.1. Preclinical studies*

*2.1.1.2. Intestinal inflammation*

*2.1.1.2.1. Preclinical studies*

**Figure 1.** Pomegranate tree.

**Figure 2.** Pomegranate fruit.

### **2. Clinical applications**

For its multiple pharmacological potential, pomegranate has been investigated by variable preclinical and clinical studies in a wide variety of health disorders:

#### **2.1. Inflammation**

Pomegranate exhibits a potent anti-inflammatory effect through inhibition of cyclooxygenase (COX) and lipoxygenase (important inflammatory mediators) [7].

#### *2.1.1. Gastrointestinal inflammation*

#### *2.1.1.1. Gastric inflammation*

#### *2.1.1.1.1. Preclinical studies*

*Helicobacter pylori* (*H. pylori)* are major etiological agents in peptic ulcer. Pomegranate methanol extract produced a remarkable anti-*H. pylori* activity with mean diameter of inhibition zone 39 at 100 μg disc−1 [8]. This activity is explained by altering bacterial cell surface hydrophobicity and prevention of bacterial adhesion to gastric mucosa [9]. Moreover, pomegranate revealed gastroprotective potential via antioxidant mechanism in aspirin- and ethanol-induced gastric ulceration in animal models [10]. Gastroprotective property of pomegranate is attributed to its constituents (saponin, tannins, and flavonoids) as demonstrated in another study on wistar rats where oral administration (490 and 980 mg/kg body weight) of pomegranate aqueous methanolic extract significantly reduced gastric ulcer index in alcohol-, indomethacin-, and aspirininduced ulcers [11]. Tannins are high molecular weight phenolic compounds present in many plants, including pomegranate fruit pericarp (peels). These compounds have the capacity to form complexes mainly with proteins [12, 13]. Pomegranate tannins form a protective layer (tannin-protein/tannin-polysaccharide complex), upon damaged epithelial tissues, thus allowing the healing process below to occur naturally through prevention of bleeding and acceleration of ulcer healing [14, 15]. All parts of the pomegranate tree have been used as a source for tannin in the leather industry, changing animal hide into leather. About 10–25% of tannin are present in the trunk bark and were important in leather production in Morocco. In this process, collagen chains in the hide are cross-linked by tannin to give leather. The formation of various complex bonds helps the tannin-protein polymer combination [16, 17]. These facts take our attention to the Islamic advice "To *eat pomegranate with its pericarp as it is tanning for the stomach*".

#### *2.1.1.2. Intestinal inflammation*

#### *2.1.1.2.1. Preclinical studies*

**2. Clinical applications**

**Figure 2.** Pomegranate fruit.

**Figure 1.** Pomegranate tree.

128 Breeding and Health Benefits of Fruit and Nut Crops

**2.1. Inflammation**

For its multiple pharmacological potential, pomegranate has been investigated by variable

Pomegranate exhibits a potent anti-inflammatory effect through inhibition of cyclooxygenase

preclinical and clinical studies in a wide variety of health disorders:

(COX) and lipoxygenase (important inflammatory mediators) [7].

Inflammatory response is induced by transduction cascades initiated by many inflammatory mediators, that is, tumor necrosis factor α (TNF-α) and nuclear factor κB (NF-κB). Pomegranate inhibited TNF-α-induced NF-κB activation and COX-2 expression in colon cell line. This effect was highly presented by pomegranate juice compared to single constituents, that is, tannin and punicalagin. This highlights the synergism between all bioactive pomegranate compounds [18]. Prebiotics are food agents that stimulate the growth or activity of beneficial microorganisms. Pomegranate peel extract (6 mg/d for 4 weeks) increased the cecal pool of beneficial bifidobacteria when given to high-fat diet mice. Additionally, it counteracted the high-fat-induced expression of inflammatory markers both in the colon and in the visceral adipose tissue [19]. Through its antioxidative action, pomegranate elagic acid (EA) (10 mg/kg) in colonic-delivering microsphere significantly ameliorated the severity of colonic lesions and reduced myeloperoxidase (MPO) activity and lipid peroxidation. This effect was obtained by orally administrating it to rat model of dextran sulfate sodium (DSS)-induced ulcerative colitis [20]. Mast cells are important inflammatory cells that release histamine. Mast cell stabilizing is an additional antiinflammatory mechanism of pomegranate where its hydroalcoholic extract significantly lowered DSS-induced elevated histamine level in mice colon tissue [21].

inflammatory changes in pulmonary tissue via its antioxidative capacity in a study that was carried out on 27 streptozotocin-induced diabetic rats, which were given either pomegranate

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 131

Prostate cancer suppression was exerted by different pomegranate fruit parts (juice, peel, and seed oil) on LNCaP, PC-3, and DU 145 human cancer cell lines. This effect was manifested by inhibition of proliferation, invasion, phosholipase A2 (PLA2) expression, and apoptosis induction [29, 30]. Pomegranate fruit extract inhibited cell growth and induced apoptosis via remodeling of apoptosis regulating proteins in prostate cancer PC-3 cell line. In addition, oral administration of pomegranate fruit extract to mice implanted with CWR22Rnu1 cells significantly suppressed tumor growth and decreased prostate-specific antigen (PSA) in the serum [31, 32]. Oral pomegranate fruit extract (100 mg/kg) for 4 weeks inhibited testosteroneinduced prostatic hyperplasia, prostate weight, prostatic acid phosphatase activity, and total

A two-stage phase-II clinical trial on 46 subjects with recurrent prostate cancer and rising serum prostate-specific antigen (PSA) after surgery or radiotherapy was carried out. The participants consumed daily eight ounces of pomegranate juice (570 mg of total polyphenol gallic acid equivalents) until meeting the disease progression endpoints. About 35% of patients achieved a significant decrease in serum (PSA). There was a significant increase in mean PSA doubling time from baseline of 15–54 months post-treatment. In a parallel in vitro study of patients' serum on LNCaP cell growth, there was a significant reduction in cell proliferation

Pomegranate constituents have been proved to be antiproliferative, noninvasive [35], apoptotic [36] angiogenesis [37], and tumor growth inhibitors [38]. Pomegranate seed oil and fermented juice polyphenols exhibited antiangiogenesis potential by suppression of vascular endothelial growth factor in MCF-10A and MCF-7 and upregulated migration inhibitory fac-

Pomegranate juice derived ellagitannins and their intestinal bacterial metabolites, urolithins, exhibited dose- and time-dependent decreases in cell proliferation, and clonogenic efficiency of HT-29 cells. The half maximal inhibitory concentration, IC50 values, ranged from 56.7 μM

and induction of apoptosis after treatment with pomegranate juice [34].

tor (MIF) in MDA-MB-231 breast cancer cell lines [38].

for urolithin A to 74.8 μM for urolithin C [39].

or saline for 10 weeks [28].

**2.2. Cancer**

*2.2.1. Prostate cancer*

*2.2.1.1. Preclinical studies*

glutathione in rats [33].

*2.2.1.2. Clinical studies*

*2.2.2. Breast cancer*

*2.2.3. Colon cancer*

#### *2.1.1.2.2. Clinical studies*

The only human trial is the ongoing phase I study on the role of pomegranate juice ellagitannins in the modulation of inflammation in inflammatory bowel diseases. This has been registered since December 2016. Available online: http://www.clinicaltrials.gov

#### *2.1.2. Joint inflammation*

#### *2.1.2.1. Preclinical studies*

A pomegranate compound, delphinidin, attenuated the inflammatory signaling that results in rheumatoid arthritis. This mechanism was mediated by inhibition of the histone acetyl transferase and NF-κB activation in human rheumatoid arthritis synovial cell line [22]. Pomegranate alleviated features of arthritis in collagen-induced arthritic mice (CIA). This effect was associated with histopathological evidence of reduced inflammatory cells and joint tissue damage. Moreover, pomegranate decreased the interleukin 6 (IL-6) level and suppressed inflammatory signal transduction pathways in mouse macrophages [23].

#### *2.1.2.2. Clinical studies*

Pomegranate (2 capsules of 250 mg pomegranate extract/day for 8 weeks) improved disease activity, some inflammatory blood biomarkers and oxidative stress (increased glutathione peroxidise) in 30 rheumatoid arthritis patients in a double-blind, placebo-controlled, randomized study [24].

#### *2.1.3. Respiratory inflammation*

#### *2.1.3.1. Preclinical studies*

Pomegranate peel aqueous extract attenuated lipopolysaccharide (LPS)-induced lung inflammation in mice. Furthermore, it inhibited the production of human neutrophil reactive oxygen species (ROS) and myeloperoxidase [25]. Synergistic anti-inflammatory effect of pomegranate extract (encapsulated into microparticles) with dexamethasone was demonstrated in asthma model mice. The microparticles attenuated leukocytes' recruitment to bronchoalveolar fluid, particularly eosinophils, reduced cytokines (IL-1β and IL-5), and reduced protein levels in the lungs. These findings supported the alternative/complementary use of pomegranate in treatment of lung inflammation [26]. Pomegranate (80 μmol/kg/day) significantly attenuated the expression of inflammatory mediators, apoptosis, and oxidative stress that were induced by acute mice exposure to cigarette smoke (for 3 days). Additionally, on chronic cigarette smoke exposure (1–3 months) pomegranate reduced expression of TNF-α and normalized lung cell architectures. Moreover, pomegranate juice attenuated the damaging effects of cigarette smoke extract on cultured human alveolar cells [27]. Pomegranate juice diminished inflammatory changes in pulmonary tissue via its antioxidative capacity in a study that was carried out on 27 streptozotocin-induced diabetic rats, which were given either pomegranate or saline for 10 weeks [28].

#### **2.2. Cancer**

important inflammatory cells that release histamine. Mast cell stabilizing is an additional antiinflammatory mechanism of pomegranate where its hydroalcoholic extract significantly low-

The only human trial is the ongoing phase I study on the role of pomegranate juice ellagitannins in the modulation of inflammation in inflammatory bowel diseases. This has been regis-

A pomegranate compound, delphinidin, attenuated the inflammatory signaling that results in rheumatoid arthritis. This mechanism was mediated by inhibition of the histone acetyl transferase and NF-κB activation in human rheumatoid arthritis synovial cell line [22]. Pomegranate alleviated features of arthritis in collagen-induced arthritic mice (CIA). This effect was associated with histopathological evidence of reduced inflammatory cells and joint tissue damage. Moreover, pomegranate decreased the interleukin 6 (IL-6) level and sup-

Pomegranate (2 capsules of 250 mg pomegranate extract/day for 8 weeks) improved disease activity, some inflammatory blood biomarkers and oxidative stress (increased glutathione peroxidise) in 30 rheumatoid arthritis patients in a double-blind, placebo-controlled, random-

Pomegranate peel aqueous extract attenuated lipopolysaccharide (LPS)-induced lung inflammation in mice. Furthermore, it inhibited the production of human neutrophil reactive oxygen species (ROS) and myeloperoxidase [25]. Synergistic anti-inflammatory effect of pomegranate extract (encapsulated into microparticles) with dexamethasone was demonstrated in asthma model mice. The microparticles attenuated leukocytes' recruitment to bronchoalveolar fluid, particularly eosinophils, reduced cytokines (IL-1β and IL-5), and reduced protein levels in the lungs. These findings supported the alternative/complementary use of pomegranate in treatment of lung inflammation [26]. Pomegranate (80 μmol/kg/day) significantly attenuated the expression of inflammatory mediators, apoptosis, and oxidative stress that were induced by acute mice exposure to cigarette smoke (for 3 days). Additionally, on chronic cigarette smoke exposure (1–3 months) pomegranate reduced expression of TNF-α and normalized lung cell architectures. Moreover, pomegranate juice attenuated the damaging effects of cigarette smoke extract on cultured human alveolar cells [27]. Pomegranate juice diminished

pressed inflammatory signal transduction pathways in mouse macrophages [23].

ered DSS-induced elevated histamine level in mice colon tissue [21].

tered since December 2016. Available online: http://www.clinicaltrials.gov

*2.1.1.2.2. Clinical studies*

130 Breeding and Health Benefits of Fruit and Nut Crops

*2.1.2. Joint inflammation*

*2.1.2.1. Preclinical studies*

*2.1.2.2. Clinical studies*

*2.1.3. Respiratory inflammation*

*2.1.3.1. Preclinical studies*

ized study [24].

#### *2.2.1. Prostate cancer*

#### *2.2.1.1. Preclinical studies*

Prostate cancer suppression was exerted by different pomegranate fruit parts (juice, peel, and seed oil) on LNCaP, PC-3, and DU 145 human cancer cell lines. This effect was manifested by inhibition of proliferation, invasion, phosholipase A2 (PLA2) expression, and apoptosis induction [29, 30]. Pomegranate fruit extract inhibited cell growth and induced apoptosis via remodeling of apoptosis regulating proteins in prostate cancer PC-3 cell line. In addition, oral administration of pomegranate fruit extract to mice implanted with CWR22Rnu1 cells significantly suppressed tumor growth and decreased prostate-specific antigen (PSA) in the serum [31, 32]. Oral pomegranate fruit extract (100 mg/kg) for 4 weeks inhibited testosteroneinduced prostatic hyperplasia, prostate weight, prostatic acid phosphatase activity, and total glutathione in rats [33].

#### *2.2.1.2. Clinical studies*

A two-stage phase-II clinical trial on 46 subjects with recurrent prostate cancer and rising serum prostate-specific antigen (PSA) after surgery or radiotherapy was carried out. The participants consumed daily eight ounces of pomegranate juice (570 mg of total polyphenol gallic acid equivalents) until meeting the disease progression endpoints. About 35% of patients achieved a significant decrease in serum (PSA). There was a significant increase in mean PSA doubling time from baseline of 15–54 months post-treatment. In a parallel in vitro study of patients' serum on LNCaP cell growth, there was a significant reduction in cell proliferation and induction of apoptosis after treatment with pomegranate juice [34].

#### *2.2.2. Breast cancer*

Pomegranate constituents have been proved to be antiproliferative, noninvasive [35], apoptotic [36] angiogenesis [37], and tumor growth inhibitors [38]. Pomegranate seed oil and fermented juice polyphenols exhibited antiangiogenesis potential by suppression of vascular endothelial growth factor in MCF-10A and MCF-7 and upregulated migration inhibitory factor (MIF) in MDA-MB-231 breast cancer cell lines [38].

#### *2.2.3. Colon cancer*

Pomegranate juice derived ellagitannins and their intestinal bacterial metabolites, urolithins, exhibited dose- and time-dependent decreases in cell proliferation, and clonogenic efficiency of HT-29 cells. The half maximal inhibitory concentration, IC50 values, ranged from 56.7 μM for urolithin A to 74.8 μM for urolithin C [39].

#### *2.2.4. Hepatocellular carcinoma*

Oxidative stress is a precipitating factor of hepatocellular carcinoma (HCC), one of the most lethal cancers. Pomegranate emulsion (1 or 10 g/kg) was given 4 weeks before dietary carcinogen diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis and 18 weeks thereafter. Pomegranate revealed chemopreventive activity manifested by reduced incidence, number, multiplicity, size, and volume of hepatic nodules. This effect was mediated by pomegranate antioxidant activity and inhibition of nuclear factor-kappaB (NF-κB) (a potent stimulant of Wnt/β catenin signaling which is involved in cell proliferation, cell survival, and apoptosis) [40, 41].

to streptozotocin-induced diabetic mice fed with a high-fat diet. Furthermore, pomegranate reduced blood glucose level and body weight [51]. Metabolic syndrome includes common clinical disorders such as obesity, hypertension, dislipidemia, and diabetes. Pomegranate juice and fruit extract induced a significant decrease in vascular inflammation markers; thrombospondin (TSP), and cytokine TGFβ1 and increase in plasma nitrate, nitrite levels, and nitric oxide-synthase expression (important factors for arterial function enhancement) in a metabolic syndrome rat model [52]. Pomegranate extract (300 mg/kg/day for 8 weeks) reduced the levels of high-fat diet-induced elevated serum interleukin 6 (IL6) and corticosterone in rats [53]. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world [54]. The pathogenesis of NAFLD includes the increased accumulation of triglyceride in hepatocytes, which progresses to nonalcoholic steatohepatitis (NASH) due to oxidative stress. In high-fat, high-sugar-diet-fed rats, pomegranate juice (60 ± 5 ml /day for 7 weeks) exhibited a significant modulation in hepatic steatosis, ballooning, lobular and portal inflammation, as well as significant attenuation of hepatic pro-inflammatory and pro-fibrotic gene expression. It significantly decreased plasma levels of alanine, aspartate aminotransferase, insulin, triglycerides, and glucose with respect to control [55]. A study comparing the antidiabetogenic effect of glibenclamide (5 mg/kg) and pomegranate juice (1 ml/day) was carried out on 40 streptozotocin (STZ)-nicotinamide (NAD)-induced type 2 diabetes mellitus rats for 21 days. Pomegranate juice (1 mL/day) showed significant repair and restoration signs in islets of Langerhans. Additionally, it significantly lowered the level of plasma total cholesterol, triglyceride, and inflammatory biomarkers, which were actively raised in diabetic rats [56].

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 133

Concentrated pomegranate juice (50 g daily for 4 weeks) exerted a significant increase in total and high-density lipoprotein cholesterol from baseline levels in 40 type 2 diabetic patients. Only serum interleukin-6 (IL-6) was significantly reduced among other tested inflammatory markers. There was about 75% increase in mean value of serum total antioxidant capacity (TAC) [57]. In a double-blinded, randomized crossover controlled study, daily 500 mL of pomegranate juice was introduced to 30 individuals with a metabolic syndrome for a week. Systolic and diastolic blood pressure as well as high sensitivity C-reactive protein was significantly reduced. However, pomegranate consumption significantly increased the level of triglyceride and low-density lipoprotein cholesterol which is attributed by the authors to the more lipogenic effect of fructose than glucose after hepatic metabolism into triglycerides [58]. On the other hand, administration of 400 mg of pomegranate seed oil capsules twice daily for 4 weeks to 25 dyslipidemic patients insignificantly reduced serum of TNF-α level [59].

Antimicrobial activity of pomegranate has been widely investigated in many studies. *Escherichia coli* (*E. coli* O157:H7) is associated with many disorders: diarrhea, hemorrhagic colitis, thrombocytopenic purpura, and hemolytic uremic syndrome. Pomegranate ethanolic

*2.4.2. Clinical studies*

**2.5. Infections**

*2.5.1. Bacterial and fungal infection*

*2.5.1.1. Preclinical studies*

#### *2.2.5. Bladder cancer*

Transitional cell carcinoma results in most of the bladder tumors [42]. The tumor suppressor gene p53 which is essential for cell cycle arrest and apoptosis [43] was believed to be inactivated in more than 50% of carcinogenesis of bladder cancers [44]. Polyphenols in pomegranate rind extract was shown to inhibit bladder cancer cell EJ proliferation via p53/miR-34a axis [45].

#### **2.3. Cardio vascular disorders**

#### *2.3.1. Preclinical studies*

Pomegranate protected against cardiovascular injury initiated by cigarette smoking in rats through its antioxidative property [46]. Moreover, antioxidative and anti-inflammatory effects of pomegranate extract reduced the size of atherosclerotic plaques in the aortic sinus and reduced the proportion of coronary arteries with occlusive atherosclerotic plaques when it was given orally in a dose of 307.5 μl/L of drinking water/day for 2 weeks to mice model of coronary heart disease [47]. Furthermore, pomegranate extract supplementation (625 mg/day) for 10 days to pigs prevented hyperlipemia-induced coronary endothelial dysfunction via a stimulation of the Akt/endothelial nitric oxide-synthase pathway [48].

#### *2.3.2. Clinical studies*

Natural pomegranate juice (150 ml/day) succeeded to significantly lower systolic and diastolic blood pressure 4–6 h post-consumption in 13 hypertensive patients [49]. Furthermore, a 1 year consumption of pomegranate juice by 10 atherosclerotic patients with carotid artery stenosis significantly reduced common carotid intima-media thickness (IMT), systolic blood pressure, and serum lipid peroxidation. Whereas after 3 years of pomegranate consumption, no additional beneficial effects occurred except for further reduction of serum lipid peroxidation by up to 16% [50].

#### **2.4. Metabolic disorders**

#### *2.4.1. Preclinical studies*

High level of low-density lipoprotein (LDL) is a risk factor for cardiovascular disease. The esterase paraoxonase1 (PON1) prevents oxidation of LDL. Decreased levels of PON1 increase the incidence of cardiovascular disease. Pomegranate juice (12.5 mL/L of juice in 1 l of water/ day for 4 months) significantly induced PON1 gene expression and activity when given daily to streptozotocin-induced diabetic mice fed with a high-fat diet. Furthermore, pomegranate reduced blood glucose level and body weight [51]. Metabolic syndrome includes common clinical disorders such as obesity, hypertension, dislipidemia, and diabetes. Pomegranate juice and fruit extract induced a significant decrease in vascular inflammation markers; thrombospondin (TSP), and cytokine TGFβ1 and increase in plasma nitrate, nitrite levels, and nitric oxide-synthase expression (important factors for arterial function enhancement) in a metabolic syndrome rat model [52]. Pomegranate extract (300 mg/kg/day for 8 weeks) reduced the levels of high-fat diet-induced elevated serum interleukin 6 (IL6) and corticosterone in rats [53]. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world [54]. The pathogenesis of NAFLD includes the increased accumulation of triglyceride in hepatocytes, which progresses to nonalcoholic steatohepatitis (NASH) due to oxidative stress. In high-fat, high-sugar-diet-fed rats, pomegranate juice (60 ± 5 ml /day for 7 weeks) exhibited a significant modulation in hepatic steatosis, ballooning, lobular and portal inflammation, as well as significant attenuation of hepatic pro-inflammatory and pro-fibrotic gene expression. It significantly decreased plasma levels of alanine, aspartate aminotransferase, insulin, triglycerides, and glucose with respect to control [55]. A study comparing the antidiabetogenic effect of glibenclamide (5 mg/kg) and pomegranate juice (1 ml/day) was carried out on 40 streptozotocin (STZ)-nicotinamide (NAD)-induced type 2 diabetes mellitus rats for 21 days. Pomegranate juice (1 mL/day) showed significant repair and restoration signs in islets of Langerhans. Additionally, it significantly lowered the level of plasma total cholesterol, triglyceride, and inflammatory biomarkers, which were actively raised in diabetic rats [56].

#### *2.4.2. Clinical studies*

*2.2.4. Hepatocellular carcinoma*

132 Breeding and Health Benefits of Fruit and Nut Crops

*2.2.5. Bladder cancer*

**2.3. Cardio vascular disorders**

*2.3.1. Preclinical studies*

*2.3.2. Clinical studies*

**2.4. Metabolic disorders**

*2.4.1. Preclinical studies*

Oxidative stress is a precipitating factor of hepatocellular carcinoma (HCC), one of the most lethal cancers. Pomegranate emulsion (1 or 10 g/kg) was given 4 weeks before dietary carcinogen diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis and 18 weeks thereafter. Pomegranate revealed chemopreventive activity manifested by reduced incidence, number, multiplicity, size, and volume of hepatic nodules. This effect was mediated by pomegranate antioxidant activity and inhibition of nuclear factor-kappaB (NF-κB) (a potent stimulant of Wnt/β catenin signaling which is involved in cell proliferation, cell survival, and apoptosis) [40, 41].

Transitional cell carcinoma results in most of the bladder tumors [42]. The tumor suppressor gene p53 which is essential for cell cycle arrest and apoptosis [43] was believed to be inactivated in more than 50% of carcinogenesis of bladder cancers [44]. Polyphenols in pomegranate rind extract was shown to inhibit bladder cancer cell EJ proliferation via p53/miR-34a axis [45].

Pomegranate protected against cardiovascular injury initiated by cigarette smoking in rats through its antioxidative property [46]. Moreover, antioxidative and anti-inflammatory effects of pomegranate extract reduced the size of atherosclerotic plaques in the aortic sinus and reduced the proportion of coronary arteries with occlusive atherosclerotic plaques when it was given orally in a dose of 307.5 μl/L of drinking water/day for 2 weeks to mice model of coronary heart disease [47]. Furthermore, pomegranate extract supplementation (625 mg/day) for 10 days to pigs prevented hyperlipemia-induced coronary endothelial dysfunction via a

Natural pomegranate juice (150 ml/day) succeeded to significantly lower systolic and diastolic blood pressure 4–6 h post-consumption in 13 hypertensive patients [49]. Furthermore, a 1 year consumption of pomegranate juice by 10 atherosclerotic patients with carotid artery stenosis significantly reduced common carotid intima-media thickness (IMT), systolic blood pressure, and serum lipid peroxidation. Whereas after 3 years of pomegranate consumption, no additional beneficial effects occurred except for further reduction of serum lipid peroxidation by up to 16% [50].

High level of low-density lipoprotein (LDL) is a risk factor for cardiovascular disease. The esterase paraoxonase1 (PON1) prevents oxidation of LDL. Decreased levels of PON1 increase the incidence of cardiovascular disease. Pomegranate juice (12.5 mL/L of juice in 1 l of water/ day for 4 months) significantly induced PON1 gene expression and activity when given daily

stimulation of the Akt/endothelial nitric oxide-synthase pathway [48].

Concentrated pomegranate juice (50 g daily for 4 weeks) exerted a significant increase in total and high-density lipoprotein cholesterol from baseline levels in 40 type 2 diabetic patients. Only serum interleukin-6 (IL-6) was significantly reduced among other tested inflammatory markers. There was about 75% increase in mean value of serum total antioxidant capacity (TAC) [57]. In a double-blinded, randomized crossover controlled study, daily 500 mL of pomegranate juice was introduced to 30 individuals with a metabolic syndrome for a week. Systolic and diastolic blood pressure as well as high sensitivity C-reactive protein was significantly reduced. However, pomegranate consumption significantly increased the level of triglyceride and low-density lipoprotein cholesterol which is attributed by the authors to the more lipogenic effect of fructose than glucose after hepatic metabolism into triglycerides [58]. On the other hand, administration of 400 mg of pomegranate seed oil capsules twice daily for 4 weeks to 25 dyslipidemic patients insignificantly reduced serum of TNF-α level [59].

#### **2.5. Infections**

#### *2.5.1. Bacterial and fungal infection*

#### *2.5.1.1. Preclinical studies*

Antimicrobial activity of pomegranate has been widely investigated in many studies. *Escherichia coli* (*E. coli* O157:H7) is associated with many disorders: diarrhea, hemorrhagic colitis, thrombocytopenic purpura, and hemolytic uremic syndrome. Pomegranate ethanolic extract was shown to be bacteriostatic and bacteriocidal against *E. coli* with minimal inhibitory concentration (MICs) from 0.49 to 1.95 mg/ml and minimal bactericidal concentration (MBCs) from 1.95 to 3.91 mg/ml [60]. Tuberculosis is an infectious disease with a time long emergence of drug resistance. Pomegranate *juice*, and *peel extracts* prepared with methanol/water, and its polyphenolic byproducts namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, were examined against drug-resistant clinical isolates of *Mycobacterium tuberculosis* and β-lactamase producing *Klebsiella pneumoniae*. The peel extracts exerted higher antimycobacterial activity (MIC 64–1024 μg/mL) than the juice (MIC 256 - > 1024 μg/mL). EGCG and quercetin showed more antitubercular and antibacterial activity than caffeic acid and ellagic acid [61]. Biofilm is a protective layer made of extracellular polymeric substances where the pathogen hides with subsequent modulation of its virulence and pathogenicity. Pomegranate methanolic extract was believed to counteract the formation of biofilms by *Staphylococcus aureus*, methicillin-resistant *S. aureus, E. coli*, and *Candida albicans*. Moreover, pomegranate extract disrupted the preformed biofilms with inhibition of germ tube formation, a virulence trait, in *C. albicans*. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75 μg ml−1) and biofilm formation at lower concentrations (<40 μg ml−1) [62]. Besides single antifungal activity, pomegranate extract showed a synergistic effect with other antimicrobial agents. Punicalagin synergism with fluconazole against *C. albicans* and *C. parapsilosis* was demonstrated in an in vitro study with a twofold decrease of MIC for fluconazole [63]. Pomegranate methanolic extract showed synergistic effect with five antibiotics: chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin against methicillin-resistant *S. aureus* strains (MRSA) and methicillin-sensitive *S. aureus* (MSSA). Most potent synergism was noticed when pomegranate was combined with ampicillin. This combination increased the post-antibiotic effect (PAE) of ampicillin from 3 to 7 h. as well as it reduced cell viability by 99.9 and 72.5% in MSSA and MRSA populations, respectively [64]. Methanol extract of pomegranate showed a synergistic action with ciprofloxacin against extended-spectrum β-lactamase (ESBL) producing *E. coli* and metallo-β-lactamase (MBL) producing *Pseudomonas aeruginosa;* that effect was attributed to bacterial efflux pump inhibitor (EPI) activity of the pomegranate polyphenolic constituents [65]. In an ongoing study of our work where we are testing the antifungal activity of some medicinal herbs against clinical isolates of *C. albicans* strain, pomegranate methanolic extract showed an inhibitory zone of 12 mm and synergistically augmented the action of fluconazole by increasing the inhibition zone from 25 to 35 mm after combination. The potent antifungal action of pomegranate led some researchers [66] to design a new antifungal peptide, pomegranin, with an N-terminal sequence from fresh pomegranate peels by ion exchange chromatography. Pomegranin suppressed mycelial growth in the fungi *Botrytis cinerea* and *Fusarium oxysporum* with half maximal inhibitory concentration (IC50) of 2 and 6.1 μM, respectively.

punicalagin), punicalagin had the highest affect against influenza A virus through suppression of viral RNA replication and agglutination of chicken RBCs. In addition, pomegranate polyphenol extract augmented the anti-influenza effect of oseltamivir when given together [67]. Pomegranate juice prevented HIV-1 binding to CD4 and blocked viral entry [68]. Moreover, agents present in pomegranate juice (polyphenols, beta-sitosterol, sugars, and ellagic acid) and fulvic acid were demonstrated as envelope virus neutralizing compounds that neutralize the viral infectivity by binding to the envelope lipid or sugar moieties [69]. Adenoviruses are a group of non-enveloped viruses that give rise to in a wide range of illnesses. Pomegranate peel ethanol extract exhibited anti-adenovirus activity on HeLa cell line where the half maximal inhibitory concentration (IC50) and 50% cytotoxicity concentration (CC50) of the extract were 165 ± 10.1 and 18.6 ± 6.7 μg/ml, respectively. The selectivity index (SI), the ratio of CC50 and IC50, was 8.89 [70]. Moreover, pomegranate tannins were shown to have anti-HSV-1, HSV-2 effect via blocking of virus adsorption to African green monkey kidney and human adenocarcinoma cells [71]. Hepatitis C virus (HCV) is the leading cause of end-stage liver disease. Ellagitannins from pomegranate peel crude extract, punicalagin, punicalin, and ellagic acid, specifically blocked the HCV NS3/4A protease activity in an in vitro study. Furthermore, punicalagin and punicalin significantly suppressed HCV replication in cell culture system. Moreover, these compounds arewere well tolerated ex vivo and "no-observed adverse effect level" (NOAEL) was established up to an acute dose of 5000 mg/kg in BALB/c mice.

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 135

Additionally, these components were bio-available by pharmacokinetics study [72].

*From ancient times,* pomegranate was described as an antihelminthic agent. Malarial infection represents a public health and economic burden in tropical and subtropical regions of the world [73]. Pomegranate gallagic acid and punicalagin exerted an antiplasmodial activity against *Plasmodium falciparum* D6 and W2 clones with IC50 values of 10.9, 10.6, 7.5, and

*Schistosomiasis* is a morbid widely distributed tropical disease [75]. Blood flukes of the genus *Schistosoma* pass a complex life cycle including multiple morphologically distinct phenotypes in definitive human and intermediate snail hosts [76]. In vitro and in vivo studies were designed to evaluate pomegranate impact on *Schistosoma mansoni* (*S. mansoni),* one of the three major species infecting humans. Pomegranate peels and leaves extracts significantly affected both adult *S. mansoni* worms and schistosomules with 100% death rate, after 24 h of exposure to plant extracts. Oral administration of the pomegranate extract to mice at a dose of 800 mg/ kg, 45 days post-infection and on three consecutive days yielded a high percentage of dead adult worms (77.30 and 72.2) with either leaves or peels extract, respectively. In addition, reduction in tissue egg load, liver, and intestinal ova counts was observed. This antiparasitic effect was confirmed by electron microscopic examination that revealed ultrastructural alterations in the tegument and the male genital systems of the worms. Bone marrow examination of pomegranate-treated *S. mansoni-*infected mice showed eosinophilic degranulation that

*2.5.3. Parasitic infection*

*2.5.3.1. Preclinical studies*

8.8 μM, respectively [74].

indicates reduced *S. mansoni* activity [77].

#### *2.5.2. Virus infection*

#### *2.5.2.1. Preclinical studies*

Pomegranate showed antiviral action against many viruses: influenza, human immuonodeficiency virus (HIV), herpes simplex virus (HSV), and adenoviruses in multiple studies. Of pomegranate polyphenol extract (PPE) constituents (ellagic acid, caffeic acid, luteolin, and punicalagin), punicalagin had the highest affect against influenza A virus through suppression of viral RNA replication and agglutination of chicken RBCs. In addition, pomegranate polyphenol extract augmented the anti-influenza effect of oseltamivir when given together [67]. Pomegranate juice prevented HIV-1 binding to CD4 and blocked viral entry [68]. Moreover, agents present in pomegranate juice (polyphenols, beta-sitosterol, sugars, and ellagic acid) and fulvic acid were demonstrated as envelope virus neutralizing compounds that neutralize the viral infectivity by binding to the envelope lipid or sugar moieties [69]. Adenoviruses are a group of non-enveloped viruses that give rise to in a wide range of illnesses. Pomegranate peel ethanol extract exhibited anti-adenovirus activity on HeLa cell line where the half maximal inhibitory concentration (IC50) and 50% cytotoxicity concentration (CC50) of the extract were 165 ± 10.1 and 18.6 ± 6.7 μg/ml, respectively. The selectivity index (SI), the ratio of CC50 and IC50, was 8.89 [70]. Moreover, pomegranate tannins were shown to have anti-HSV-1, HSV-2 effect via blocking of virus adsorption to African green monkey kidney and human adenocarcinoma cells [71]. Hepatitis C virus (HCV) is the leading cause of end-stage liver disease. Ellagitannins from pomegranate peel crude extract, punicalagin, punicalin, and ellagic acid, specifically blocked the HCV NS3/4A protease activity in an in vitro study. Furthermore, punicalagin and punicalin significantly suppressed HCV replication in cell culture system. Moreover, these compounds arewere well tolerated ex vivo and "no-observed adverse effect level" (NOAEL) was established up to an acute dose of 5000 mg/kg in BALB/c mice. Additionally, these components were bio-available by pharmacokinetics study [72].

#### *2.5.3. Parasitic infection*

extract was shown to be bacteriostatic and bacteriocidal against *E. coli* with minimal inhibitory concentration (MICs) from 0.49 to 1.95 mg/ml and minimal bactericidal concentration (MBCs) from 1.95 to 3.91 mg/ml [60]. Tuberculosis is an infectious disease with a time long emergence of drug resistance. Pomegranate *juice*, and *peel extracts* prepared with methanol/water, and its polyphenolic byproducts namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, were examined against drug-resistant clinical isolates of *Mycobacterium tuberculosis* and β-lactamase producing *Klebsiella pneumoniae*. The peel extracts exerted higher antimycobacterial activity (MIC 64–1024 μg/mL) than the juice (MIC 256 - > 1024 μg/mL). EGCG and quercetin showed more antitubercular and antibacterial activity than caffeic acid and ellagic acid [61]. Biofilm is a protective layer made of extracellular polymeric substances where the pathogen hides with subsequent modulation of its virulence and pathogenicity. Pomegranate methanolic extract was believed to counteract the formation of biofilms by *Staphylococcus aureus*, methicillin-resistant *S. aureus, E. coli*, and *Candida albicans*. Moreover, pomegranate extract disrupted the preformed biofilms with inhibition of germ tube formation, a virulence trait, in *C. albicans*. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75 μg ml−1) and biofilm formation at lower concentrations (<40 μg ml−1) [62]. Besides single antifungal activity, pomegranate extract showed a synergistic effect with other antimicrobial agents. Punicalagin synergism with fluconazole against *C. albicans* and *C. parapsilosis* was demonstrated in an in vitro study with a twofold decrease of MIC for fluconazole [63]. Pomegranate methanolic extract showed synergistic effect with five antibiotics: chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin against methicillin-resistant *S. aureus* strains (MRSA) and methicillin-sensitive *S. aureus* (MSSA). Most potent synergism was noticed when pomegranate was combined with ampicillin. This combination increased the post-antibiotic effect (PAE) of ampicillin from 3 to 7 h. as well as it reduced cell viability by 99.9 and 72.5% in MSSA and MRSA populations, respectively [64]. Methanol extract of pomegranate showed a synergistic action with ciprofloxacin against extended-spectrum β-lactamase (ESBL) producing *E. coli* and metallo-β-lactamase (MBL) producing *Pseudomonas aeruginosa;* that effect was attributed to bacterial efflux pump inhibitor (EPI) activity of the pomegranate polyphenolic constituents [65]. In an ongoing study of our work where we are testing the antifungal activity of some medicinal herbs against clinical isolates of *C. albicans* strain, pomegranate methanolic extract showed an inhibitory zone of 12 mm and synergistically augmented the action of fluconazole by increasing the inhibition zone from 25 to 35 mm after combination. The potent antifungal action of pomegranate led some researchers [66] to design a new antifungal peptide, pomegranin, with an N-terminal sequence from fresh pomegranate peels by ion exchange chromatography. Pomegranin suppressed mycelial growth in the fungi *Botrytis cinerea* and *Fusarium oxysporum* with half maximal inhibitory concentration (IC50) of 2 and 6.1 μM, respectively.

134 Breeding and Health Benefits of Fruit and Nut Crops

Pomegranate showed antiviral action against many viruses: influenza, human immuonodeficiency virus (HIV), herpes simplex virus (HSV), and adenoviruses in multiple studies. Of pomegranate polyphenol extract (PPE) constituents (ellagic acid, caffeic acid, luteolin, and

*2.5.2. Virus infection*

*2.5.2.1. Preclinical studies*

#### *2.5.3.1. Preclinical studies*

*From ancient times,* pomegranate was described as an antihelminthic agent. Malarial infection represents a public health and economic burden in tropical and subtropical regions of the world [73]. Pomegranate gallagic acid and punicalagin exerted an antiplasmodial activity against *Plasmodium falciparum* D6 and W2 clones with IC50 values of 10.9, 10.6, 7.5, and 8.8 μM, respectively [74].

*Schistosomiasis* is a morbid widely distributed tropical disease [75]. Blood flukes of the genus *Schistosoma* pass a complex life cycle including multiple morphologically distinct phenotypes in definitive human and intermediate snail hosts [76]. In vitro and in vivo studies were designed to evaluate pomegranate impact on *Schistosoma mansoni* (*S. mansoni),* one of the three major species infecting humans. Pomegranate peels and leaves extracts significantly affected both adult *S. mansoni* worms and schistosomules with 100% death rate, after 24 h of exposure to plant extracts. Oral administration of the pomegranate extract to mice at a dose of 800 mg/ kg, 45 days post-infection and on three consecutive days yielded a high percentage of dead adult worms (77.30 and 72.2) with either leaves or peels extract, respectively. In addition, reduction in tissue egg load, liver, and intestinal ova counts was observed. This antiparasitic effect was confirmed by electron microscopic examination that revealed ultrastructural alterations in the tegument and the male genital systems of the worms. Bone marrow examination of pomegranate-treated *S. mansoni-*infected mice showed eosinophilic degranulation that indicates reduced *S. mansoni* activity [77].

#### **2.6. Central nervous system disorders**

#### *2.6.1. Cognitive disorders*

#### *2.6.1.1. Preclinical studies*

Cognitive disorders affect learning, memory, perception, and problem-solving. These disorders include amnesia, dementia, and delirium. Pomegranate ellagic acid (30 and 100 mg/kg) ameliorated scopolamine- (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.)-induced amnesia in mice. Furthermore, chronic administration of ellagic acid (30 mg/kg) improved the memory deficit induced by diazepam (1 mg/kg) in rats [78]. Memory impairment, a feature of Alzheimer's disease (AD), is initiated by neuroinflammation and impairments in synaptic plasticity. These disorders are induced by the effect of extracellular amyloid-beta (Aβ) deposits called senile plaques. The generation of Aβ is dependent on the proteolytic processing of amyloid precursor protein (APP) [79]. Pomegranate is believed to slow the rate of neurodegeneration in Alzheimer's disease. At a cellular level, pomegranate compound, punicalagin, was examined for its memory protective anti-inflammatory effect on lipopolysaccharide (LPS)-induced neuroinflammation in astrocytes and microglial BV-2 cells. In a dose of 1.5 mg/kg punicalagin attenuated LPS (250 μg/kg daily 7 times) induced memory impairment and blocked the LPS-induced expression of inflammatory proteins via suppression of NF-κB activation [80]. In addition, freeze-dried pomegranate (25– 200 μg/ml) in a dose-dependent manner reduced COX-2-dependent prostaglandin E2 (PGE2) production in SK-N-SH cells stimulated with IL-1β [81]. The neuroprotective action of pomegranate was obscured in an animal study in which dietary supplementation of 4% pomegranate extract to APPsw/Tg2576 mice for 15 months ameliorated the loss of synaptic structure proteins, inhibited neuroinflammatory activity, and enhanced autophagy (degradation and recycling of cellular components). Moreover, it reduced β-site cleavage of APP [82]. Along with figs and dates, pomegranate dietary intake attenuated the levels of inflammatory cytokines in APPsw/Tg2576 mice a model of Alzheimer disease, as well as delayed the formation of senile plaques [83].

Pomegranate seed oil (PSO) in nanodroplet formulation induced more significant beneficial effects the in mice model of multiple sclerosis (MS) than natural pomegranate seed oil. This effect was evident by dramatic alleviation of lipid demyelination and oxidation in mice brains [85].

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 137

Neonatal hypoxic-ischemic (HI) brain injury is a fatal condition that affects preterm very low birth-weight infants. After administration to pregnant mice, pomegranate juice revealed antioxidant-driven neuroprotective effect in experimentally induced HI brain injured neonatal

Prolonged human exposure to sun's ultraviolet (UV) radiation, especially its UV-B, causes many adverse effects. Pomegranate fruit extract was proved to be *a* photo-chemo preventive agent on human epidermal keratinocytes. It alleviated ultraviolet A and B radiation-induced

Oral elagic acid-rich pomegranate extract either in high (200 mg/d ellagic acid) or low doses (100 mg/d ellagic acid) improved ultraviolet-induced skin pigmentation of 26 subjects in

Pomegranate juice improved epididymal sperm concentration, spermatogenic cell density, diameter of seminiferous tubules, and sperm motility. It decreased the number of abnormal sperms compared to control rat animals. Moreover*,* pomegranate juice resulted in improvement of antioxidant enzyme activity in both rat plasma and sperm [91]. Pomegranate juice significantly increased intracavernous blood flow and smooth muscle relaxation in a rabbit

In a randomized, double-blind, placebo-controlled, 10-week crossover trial, pomegranate juice (1.5 mmol polyphenols daily) showed insignificant improvement when introduced to 53

cell damage in a dose- and time-dependent manner [88, 89].

4 weeks double-blind placebo-controlled trial [90].

*2.7.2. Male infertility and erectile dysfunction*

model of arteriogenic erectile dysfunction [92].

men with mild-to-moderate erectile dysfunction [93].

*2.6.4. Neonatal hypoxic-ischemic brain injury*

*2.6.4.1. Preclinical studies*

offsprings [86–87].

*2.7.1. Skin disorders*

*2.7.1.1. Preclinical studies*

*2.7.1.2. Clinical studies*

*2.7.2.1. Preclinical studies*

*2.7.2.2. Clinical studies*

**2.7. Miscellaneous disorders**

#### *2.6.2. Ischemic stroke*

#### *2.6.2.1. Preclinical studies*

Ischemic stroke is one of the neurodegenerative diseases. An in vitro study utilized serum glucose deprivation (SGD) as a model for ischemia-induced brain injury in PC12 cells. Pretreatment with different pomegranate extracts, namely, pulp hydroalcoholic extract (PHE), pulp aqueous extract (PAE), and pomegranate for 2 h significantly and concentration-dependently, increased cell viability and decreased DNA damage initiated by SGD insult [84].

#### *2.6.3. Multiple sclerosis*

#### *2.6.3.1. Preclinical studies*

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. Pomegranate seed oil (PSO) in nanodroplet formulation induced more significant beneficial effects the in mice model of multiple sclerosis (MS) than natural pomegranate seed oil. This effect was evident by dramatic alleviation of lipid demyelination and oxidation in mice brains [85].

#### *2.6.4. Neonatal hypoxic-ischemic brain injury*

#### *2.6.4.1. Preclinical studies*

**2.6. Central nervous system disorders**

136 Breeding and Health Benefits of Fruit and Nut Crops

Cognitive disorders affect learning, memory, perception, and problem-solving. These disorders include amnesia, dementia, and delirium. Pomegranate ellagic acid (30 and 100 mg/kg) ameliorated scopolamine- (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.)-induced amnesia in mice. Furthermore, chronic administration of ellagic acid (30 mg/kg) improved the memory deficit induced by diazepam (1 mg/kg) in rats [78]. Memory impairment, a feature of Alzheimer's disease (AD), is initiated by neuroinflammation and impairments in synaptic plasticity. These disorders are induced by the effect of extracellular amyloid-beta (Aβ) deposits called senile plaques. The generation of Aβ is dependent on the proteolytic processing of amyloid precursor protein (APP) [79]. Pomegranate is believed to slow the rate of neurodegeneration in Alzheimer's disease. At a cellular level, pomegranate compound, punicalagin, was examined for its memory protective anti-inflammatory effect on lipopolysaccharide (LPS)-induced neuroinflammation in astrocytes and microglial BV-2 cells. In a dose of 1.5 mg/kg punicalagin attenuated LPS (250 μg/kg daily 7 times) induced memory impairment and blocked the LPS-induced expression of inflammatory proteins via suppression of NF-κB activation [80]. In addition, freeze-dried pomegranate (25– 200 μg/ml) in a dose-dependent manner reduced COX-2-dependent prostaglandin E2 (PGE2) production in SK-N-SH cells stimulated with IL-1β [81]. The neuroprotective action of pomegranate was obscured in an animal study in which dietary supplementation of 4% pomegranate extract to APPsw/Tg2576 mice for 15 months ameliorated the loss of synaptic structure proteins, inhibited neuroinflammatory activity, and enhanced autophagy (degradation and recycling of cellular components). Moreover, it reduced β-site cleavage of APP [82]. Along with figs and dates, pomegranate dietary intake attenuated the levels of inflammatory cytokines in APPsw/Tg2576 mice a model of Alzheimer disease, as well as delayed the formation of senile plaques [83].

Ischemic stroke is one of the neurodegenerative diseases. An in vitro study utilized serum glucose deprivation (SGD) as a model for ischemia-induced brain injury in PC12 cells. Pretreatment with different pomegranate extracts, namely, pulp hydroalcoholic extract (PHE), pulp aqueous extract (PAE), and pomegranate for 2 h significantly and concentration-dependently, increased cell viability and decreased DNA damage initiated by SGD

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress.

*2.6.1. Cognitive disorders*

*2.6.1.1. Preclinical studies*

*2.6.2. Ischemic stroke*

insult [84].

*2.6.2.1. Preclinical studies*

*2.6.3. Multiple sclerosis*

*2.6.3.1. Preclinical studies*

Neonatal hypoxic-ischemic (HI) brain injury is a fatal condition that affects preterm very low birth-weight infants. After administration to pregnant mice, pomegranate juice revealed antioxidant-driven neuroprotective effect in experimentally induced HI brain injured neonatal offsprings [86–87].

#### **2.7. Miscellaneous disorders**

#### *2.7.1. Skin disorders*

#### *2.7.1.1. Preclinical studies*

Prolonged human exposure to sun's ultraviolet (UV) radiation, especially its UV-B, causes many adverse effects. Pomegranate fruit extract was proved to be *a* photo-chemo preventive agent on human epidermal keratinocytes. It alleviated ultraviolet A and B radiation-induced cell damage in a dose- and time-dependent manner [88, 89].

#### *2.7.1.2. Clinical studies*

Oral elagic acid-rich pomegranate extract either in high (200 mg/d ellagic acid) or low doses (100 mg/d ellagic acid) improved ultraviolet-induced skin pigmentation of 26 subjects in 4 weeks double-blind placebo-controlled trial [90].

#### *2.7.2. Male infertility and erectile dysfunction*

#### *2.7.2.1. Preclinical studies*

Pomegranate juice improved epididymal sperm concentration, spermatogenic cell density, diameter of seminiferous tubules, and sperm motility. It decreased the number of abnormal sperms compared to control rat animals. Moreover*,* pomegranate juice resulted in improvement of antioxidant enzyme activity in both rat plasma and sperm [91]. Pomegranate juice significantly increased intracavernous blood flow and smooth muscle relaxation in a rabbit model of arteriogenic erectile dysfunction [92].

#### *2.7.2.2. Clinical studies*

In a randomized, double-blind, placebo-controlled, 10-week crossover trial, pomegranate juice (1.5 mmol polyphenols daily) showed insignificant improvement when introduced to 53 men with mild-to-moderate erectile dysfunction [93].

#### *2.7.3. Dental disorders*

#### *2.7.3.1. Preclinical studies*

Bacterial and fungal co-infection initiates oral diseases. Pomegranate phytotherapeutic gel was shown to be superior to miconazole in attenuation of microbial adherence with three and four associated organisms: *Streptococci* strains (*mutans* ATCC 25175, *sanguis* ATCC 10577 and *mitis* ATCC 9811) and *C. albicans* [94]. Elagic acid exerted a moderate inhibitory effect at 12.5 mg/mL with inhibition to adherence <50% against different strains of Streptococcus mutans bacteria that induced dental caries [95].

(48 h) of urinary excreted urolithin metabolites after 180 ml of pomegranate juice consumption. Prolonged stay of urolithins in the human body is responsible for the health benefits of chronic pomegranate consumption [104]. A 1 liter pomegranate juice containing 4.37 g/L punicalagins and 0.49 g/L anthocyanins was introduced to six healthy individuals for 5 days; urolithin A, urolithin B, and a third unidentified minor metabolite were detected in plasma as well as in urine analysis at 24 h besides an aglycone metabolite corresponding to each of three plasma metabolites. Maximum excretion rates occurred 3–4 days after juice ingestion. The concentrations of urinary metabolites varied significantly in the subjects which may be attributed to colonic microflora variability and the site of ellagitannins metabolism [105]. A crossover pharmacokinetic study reported that higher free ellagic acid EA intake does not enhance its bioavailability in healthy volunteers who consumed two pomegranate extracts of 130 mg punicalagin+524 mg ellagic acid or 279 mg punicalagin+25 mg ellagic acid. The study showed high inter-individual variability; Cmax ranged from 12 to 360 nM that may be attrib-

Clinical Applications of Pomegranate http://dx.doi.org/10.5772/intechopen.75962 139

Pomegranate is safe when it is used in normal doses [107]. The median lethal dose, LD 50 of the whole fruit extract, was 731 mg/kg after intra-peritoneal administration to OF-1 mice [108]. Standardized pomegranate extract of 30% punicalagins showed acute oral LD50 in wistar rats and in Swiss albino mice it was more than 5000 mg/kg. Subchronic no-observed adverse effect level (NOAEL) was 600 mg/kg body weight/day [109]. Pomegranate ellagitannin-enriched polyphenol extract in a daily dose of 1420 mg (870 mg of gallic acid equivalents,) for 28 days

Pomegranate*'*s uncountable beneficial pharmacological properties encourage more and more

First and foremost, my deepest gratitude to GOD, for his uncountable gifts including pomegranate. Second, I would like to thank my parents and family for continuous encouragement. I would also like to express my thanks to Dr. Farid Badria, Professor of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt, and Dr. Khalil Mahfouz, Assistant Professor of Botany, Faculty of Science, Tanta University, Egypt, for their generous advice and help. My great appreciation to Professor Dr. Said Shalaby, Vice President, Academy of

Scientific Research and Technology, Cairo, Egypt, for his unforgettable support.

uted to the ellagitannin pH and protein environment [106].

showed no adverse effects in 64 overweight subjects [110].

studies to discover other secrets for solving mankind health problems.

**4. Safety**

**5. Conclusion**

**Acknowledgements**

#### *2.7.3.2. Clinical studies*

In a human study, pomegranate hydroalcoholic extract was superior to chlorhexidine (standard and positive control) in decreasing the colony forming unit (CFU)/ml by 84 and 79%, respectively, of dental plaque microorganisms [96]. Pomegranate along with *Centella asiatica* extracts significantly improved clinical signs of chronic periodontitis and IL-1beta level when it was applied with biodegradable chips on periodontal disease in 20 patients with remaining probing pocket depths after conventional periodontal therapy [97]. Pomegranate gel was compared to miconazol gel (3 times daily for 15 days) in 60 patients suffering from denture stomatitis. The patients were randomly distributed into two groups of 30 patients each. Clinical response was statistically better in miconazol group (P < 0.01) with similar fungal negativity in both groups. Clinical response and fungal negativity was achieved in 21 and 23 patients of pomegranate group as compared to 27 and 25 subjects who received miconazol, respectively. Side effects were only reported from all miconazol-treated patients. The authors explained the better miconazol clinical response by the bigger number of subjects with good oral hygiene score in the miconazol group and the longer duration of miconazol (sticky formulation) in the mouth than pomegranate gel that was washed away on mixing with saliva [98].

#### **3. Pharmacokinetic studies**

Pomegranate ellagitannins release ellagic acid in the gut, and this compound is poorly absorbed in the small intestine, while it is largely metabolized by human gut microflora into urolithins, such as urolithins A and B and urolithin-8-methyl ether in the large intestine [99]. Pomegranate anthocyanins (the 3-glucosides and 3, 5-diglucosides of delphinidin, cyanidin, and pelargonidinare) are stable in the stomach. While in the neutral pH of the small and large intestines, anthocyanins become less stable and are converted into a variety of metabolites [100–102].

The maximum plasma concentration (Cmax) of ellagic acid was 33 ng/mL and time of maximum concentration (Tmax) was 1 h [103]. A pharmacokinetic study on 18 healthy volunteers proved the rapid absorption and plasma clearance of ellagitannins as well as long persistence (48 h) of urinary excreted urolithin metabolites after 180 ml of pomegranate juice consumption. Prolonged stay of urolithins in the human body is responsible for the health benefits of chronic pomegranate consumption [104]. A 1 liter pomegranate juice containing 4.37 g/L punicalagins and 0.49 g/L anthocyanins was introduced to six healthy individuals for 5 days; urolithin A, urolithin B, and a third unidentified minor metabolite were detected in plasma as well as in urine analysis at 24 h besides an aglycone metabolite corresponding to each of three plasma metabolites. Maximum excretion rates occurred 3–4 days after juice ingestion. The concentrations of urinary metabolites varied significantly in the subjects which may be attributed to colonic microflora variability and the site of ellagitannins metabolism [105]. A crossover pharmacokinetic study reported that higher free ellagic acid EA intake does not enhance its bioavailability in healthy volunteers who consumed two pomegranate extracts of 130 mg punicalagin+524 mg ellagic acid or 279 mg punicalagin+25 mg ellagic acid. The study showed high inter-individual variability; Cmax ranged from 12 to 360 nM that may be attributed to the ellagitannin pH and protein environment [106].

### **4. Safety**

*2.7.3. Dental disorders*

*2.7.3.1. Preclinical studies*

138 Breeding and Health Benefits of Fruit and Nut Crops

*2.7.3.2. Clinical studies*

with saliva [98].

[100–102].

**3. Pharmacokinetic studies**

mutans bacteria that induced dental caries [95].

Bacterial and fungal co-infection initiates oral diseases. Pomegranate phytotherapeutic gel was shown to be superior to miconazole in attenuation of microbial adherence with three and four associated organisms: *Streptococci* strains (*mutans* ATCC 25175, *sanguis* ATCC 10577 and *mitis* ATCC 9811) and *C. albicans* [94]. Elagic acid exerted a moderate inhibitory effect at 12.5 mg/mL with inhibition to adherence <50% against different strains of Streptococcus

In a human study, pomegranate hydroalcoholic extract was superior to chlorhexidine (standard and positive control) in decreasing the colony forming unit (CFU)/ml by 84 and 79%, respectively, of dental plaque microorganisms [96]. Pomegranate along with *Centella asiatica* extracts significantly improved clinical signs of chronic periodontitis and IL-1beta level when it was applied with biodegradable chips on periodontal disease in 20 patients with remaining probing pocket depths after conventional periodontal therapy [97]. Pomegranate gel was compared to miconazol gel (3 times daily for 15 days) in 60 patients suffering from denture stomatitis. The patients were randomly distributed into two groups of 30 patients each. Clinical response was statistically better in miconazol group (P < 0.01) with similar fungal negativity in both groups. Clinical response and fungal negativity was achieved in 21 and 23 patients of pomegranate group as compared to 27 and 25 subjects who received miconazol, respectively. Side effects were only reported from all miconazol-treated patients. The authors explained the better miconazol clinical response by the bigger number of subjects with good oral hygiene score in the miconazol group and the longer duration of miconazol (sticky formulation) in the mouth than pomegranate gel that was washed away on mixing

Pomegranate ellagitannins release ellagic acid in the gut, and this compound is poorly absorbed in the small intestine, while it is largely metabolized by human gut microflora into urolithins, such as urolithins A and B and urolithin-8-methyl ether in the large intestine [99]. Pomegranate anthocyanins (the 3-glucosides and 3, 5-diglucosides of delphinidin, cyanidin, and pelargonidinare) are stable in the stomach. While in the neutral pH of the small and large intestines, anthocyanins become less stable and are converted into a variety of metabolites

The maximum plasma concentration (Cmax) of ellagic acid was 33 ng/mL and time of maximum concentration (Tmax) was 1 h [103]. A pharmacokinetic study on 18 healthy volunteers proved the rapid absorption and plasma clearance of ellagitannins as well as long persistence Pomegranate is safe when it is used in normal doses [107]. The median lethal dose, LD 50 of the whole fruit extract, was 731 mg/kg after intra-peritoneal administration to OF-1 mice [108]. Standardized pomegranate extract of 30% punicalagins showed acute oral LD50 in wistar rats and in Swiss albino mice it was more than 5000 mg/kg. Subchronic no-observed adverse effect level (NOAEL) was 600 mg/kg body weight/day [109]. Pomegranate ellagitannin-enriched polyphenol extract in a daily dose of 1420 mg (870 mg of gallic acid equivalents,) for 28 days showed no adverse effects in 64 overweight subjects [110].

### **5. Conclusion**

Pomegranate*'*s uncountable beneficial pharmacological properties encourage more and more studies to discover other secrets for solving mankind health problems.

### **Acknowledgements**

First and foremost, my deepest gratitude to GOD, for his uncountable gifts including pomegranate. Second, I would like to thank my parents and family for continuous encouragement. I would also like to express my thanks to Dr. Farid Badria, Professor of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt, and Dr. Khalil Mahfouz, Assistant Professor of Botany, Faculty of Science, Tanta University, Egypt, for their generous advice and help. My great appreciation to Professor Dr. Said Shalaby, Vice President, Academy of Scientific Research and Technology, Cairo, Egypt, for his unforgettable support.

#### **Author details**

Sally Elnawasany

Address all correspondence to: elnawasany\_s@hotmail.com

Faculty of Medicine, Tanta University, Tanta, Egypt

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## *Edited by Jaya R. Soneji and Madhugiri Nageswara-Rao*

The fruit and nut crops are laden with health benefits. As people are becoming more conscious about their health and nutritional uptake, the worldwide demand and consumption of fruit and nut crops are steadily increasing. This has made it hard to keep pace between the rate of fruit and nut production and its consumption. To meet this increasing demand, there is a need to produce improved, better yielding, and high-quality fruit and nut crops. This book intends to provide the reader with a comprehensive overview of the current status and future prospects of fruit and nut crops. Such information covered in this book will directly enhance both basic and applied research in fruit and nut crops and will particularly be useful for students, scientists, researchers, teachers, breeders, policy-makers, and growers.

Published in London, UK © 2018 IntechOpen © Hendra Su / iStock

Breeding and Health Benefits of Fruit and Nut Crops

Breeding and Health Benefits

of Fruit and Nut Crops

*Edited by Jaya R. Soneji* 

*and Madhugiri Nageswara-Rao*