Bioactive Flavonoids from Citrus Fruits

Bioactive Flavonoids from Citrus Fruits: History

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Subjects: Biochemistry & Molecular Biology

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Citrus species are one of the world’s popular fruit crops, cultivated all over the world for their economic and nutritional values. Citrus, like other fruits and vegetables, are an important source of several antioxidant molecules (polyphenols, ascorbic acid, and carotenoids) that can inhibit the harmful effects of free radicals on the human body; due to their functional values and health-promoting properties, Citrus species are considered valuable fruits not only in agri-food industry, but also in pharmaceutical industry. Flavonoids are among the major constituents of polyphenols found in different parts of Citrus fruits (skin, peels, seed, pulp membrane, and juice). Flavonoids have different biological properties (antiviral, antifungal, and antibacterial activities). Several studies have also shown the health-related properties of Citrus flavonoids, especially antioxidant, anticancer, anti-inflammation, anti-aging, and cardiovascular protection activities.

Citrus genus
bioactive molecules
flavonoids
therapeutic effects
extraction methods

1. Introduction

Citrus genus is one of the world’s leading fruit crops cultivated for food processing as well as fresh juice production. The genus Citrus belongs to the family Rutaceae, containing several species such as orange types, sweet and sour oranges, lemon, tangerines (mandarins), and tangors. Each species or hybrid cross has one or more varieties. Besides being a rich source of vitamins A, C, and E, mineral elements, and dietary fibers, Citrus fruits are a great source of secondary metabolites, such as polyphenols and terpenoids [1]. Flavonoids and phenolic acids are the main classes of phenolic compounds found in Citrus fruits [2]. In general, the fruit skin contains a higher concentration of antioxidant substances than the other parts of the fruit [3]. Citrus flavonoid contents and profiles vary significantly from one species to another [4]. Citrus peel, which represents between 50% and 65% of the total weight of the fruits, is a rich source of bioactive compounds, including natural antioxidants such as flavonoids [5]. Several studies showed that Citrus flavonoids are found to have anti-inflammatory, anti-cancer, anti-bacterial, anti-aging, and cardiovascular protection activities [6,7].

2. Citrus Flavonoids: Structure, Classification and Biosynthesis

2.1. Structure and Classification of Flavonoids from Citrus

Flavonoids are an important class of natural products; particularly, they belong to polyphenol compounds and are synthesized by plants via primary or secondary metabolisms that protect against short-term or long-term threats and play a major function in plant development and reproduction [12]. Flavonoids are found widespread throughout the plant kingdom and are associated with many health benefits [13]. They are a major class of phytochemicals discovered in citrus fruits, especially in peels, pulp, and seeds. Flavonoids are small molecular weight polyphenolic substances that have the same basic skeleton of fifteen carbons (C6-C3-C6), consisting of two phenyl rings (A and B) connected by a heterocyclic pyran or pyron ring (C) in the center, depending on their substituents. Flavonoids are subdivided into flavanols, anthocyanidins, flavanones, flavones, and chalcones [14]. The generic flavonoid structure and the numbering system used to distinguish the carbon positions around the molecule are shown in Table 2. The three phenolic rings that make up the flavonoid molecule are called pyran rings. Citrus flavonoids are divided into three principal types, namely, flavanones, flavones, and flavonols [15]. In Table 2, the classification of Citrus flavonoids and the chemical structures of major flavonoids are presented. The main flavonoids found in Citrus species are hesperidin, narirutin, naringin and eriocitrin.

Table 2. Basic structures of Citrus flavonoid subclasses [16].

Citrus Flavonoids	Examples
Flavanones	Hesperetin	R₁=O-CH₃
	Hesperetin	R₂=OH
	Naringenin	R₁=OH
	Naringenin	R₂=H
Flavanones glycosides	Narirutin	R₁=O-CH₃
		R₂=H
		R₃=7-O-rutinoside
	Naringin	R₁=OH
		R₂=H
		R₃=O-7-Rhamnoglucoside
Flavones	Apigenin	R₁=OH
	Apigenin	R₂=R₃=H
	Luteolin	R₁=R₂=OH
	Luteolin	R₃=H
Polymethoxylated Flavones	Nobiletin	R₁=H
	Nobiletin	R₂=R₃=OCH₃
	Tangeretin	R₁=R₃=H
	Tangeretin	R₂=OCH₃
Flavonols	Quercetin	R₁=R₂=R₃=OH
	Kaempferol	R₁=R₃=OH
	Kaempferol	R₂=H

2.2. Biosynthesis of Flavonoids

The flavonoid pathway is preceded by the general phenylpropanoid pathway, in which three enzymes are involved in the conversion of the amino acid phenylalanine to 4-coumaroyl-CoA. the first enzyme, phenylalanine ammonia lyase (PAL:EC 4.3.1.24) catalyzes the conversion of the amino acid phenylalanine to trans-cinnamic acid, with the release of ammonia (NH3), then the two other enzymes (the enzyme cinnamate 4-hydroxylase (C4H:EC 1.14.14.91), followed by 4-coumarate-CoA ligase (4CL:EC 6.2.1.12)), catalyze the reaction that leads to obtain 4-coumaroyl-CoA, which is an important precursor in the flavonoid pathway [12,13]. The biosynthesis of flavonoids is originated from the phenylpropanoids pathway and initiated by two precursors named malonyl-CoA and p-coumaroyl-CoA (Figure 1). After the condensation of three acetate units from malonyl-CoA with one molecule of p-coumaroyl-CoA, naringenin chalcones are formed. Naringenin chalcone, a major pigment of many flowers, leaves and fruits, is converted to naringenin by chalcone isomerase (CHI) or non-enzymatically in vitro [14,15]. This reaction catalyzed by chalcone synthase (CHS:EC 2.3.1.74) is thought to be the key regulatory step in the synthesis of flavonoids. It catalyzes the stereospecific isomerization of chalcones to their corresponding (2S)-flavanones via an acid base catalysis mechanism; the unstable chalcone form is normally isomerized by the enzyme chalcone isomerase (CHI:EC 5.5.1.6) to form the structural precursors for a broad range of flavonoids, such as flavonols, flavanones, anthocyanin glycosides and other derived compounds (Figure 1).

Figure 1. Plant flavonoids biosynthetic pathway [15]. Enzymes for each step are indicated as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; DFR, dihydro-flavonol 4-reductase; FNS, flavonol synthase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose: flavonoid-3-O-glycosyltransferase.

(2S)-Flavanones are converted stereospecifically to the respective (2R,3R)-dihydroflavonols (DHFs) by flavanone 3-hydroxylase (F3H:EC 1.14.11.9) in this case to dihydrokaempferol, then converted by the enzyme flavonoid 3′-hydroxylase (F3′H:EC 1.14.14.82) to dihydroquercetin. In Citrus species, the two dihydroflavanols are then converted into flavonols under the action of flavonols synthetase (FLS:EC 1.14.20.6). UDP-glucose flavanone-7-O-glucosyltransferase (UFGT:EC 2.4.1.115) and UDP-rhamnose flavanone glucoside rhamnosyltransferase (UFGRT) catalyze the conversion of the flavanone aglycones into their glucosides and rhamnoglucosides [16]. Dihydroflavonol-4-reductase (DFR:EC 1.1.1.219) is a key enzyme required for both anthocyanin biosynthesis and proanthocyanidin accumulation and is involved in the conversion of dihydroflavonols to leucoanthocyanidins [17].

Flavan-3-ols, such as catechin and epicatechin, are synthetized from leucoanthocyanidin and anthocyanidin. Leucoanthocyanidin can be converted to flavan-3-ol (for example, catechin) by leucoanthocyanidin reductase (LAR:EC 1.17.1.3) or to anthocyanidin by anthocyanidin synthase (ANS:EC 1.14.20.4). Anthocyanidin as well can be converted to flavan-3-ol (for example, epicatechin) by anthocyanidin reductase (ANR:EC 1.3.1.77), or glycosylated to form anthocyanin [18].

3. Flavonoid Composition in Different Parts of Citrus Fruits

It has been found that Citrus flavonoids are present in almost all the parts of Citrus fruits in different species. Several studies showed that peels are the main sources of polyphenols in Citrus fruits [19]. Seed and peel extracts from numerous Citrus species have proved to be an important source of flavonoids, such as polymethoxyflavones (PMFs), flavanones, and glycosylated flavanones [20]. More than 5000 flavonoid molecules have been identified [21]. Citrus juice is also a major source of flavanone glycosides. Moreover, the contents and types of flavonoids vary among different Citrus species and fruit parts [21]. Flavanones are the most important flavonoids in Citrus species. Hesperedin and naringenin are the predominant flavanones in Citrus fruits [16]. They can be found in all plant parts: stem, branches, bark, flowers, leaves, roots, rhizomes, seeds, fruits, peels, etc. Flavones are the second major group of flavonoids in Citrus, with flavonols in third place because their content is much lower than the other flavonoids [17,18]. The content of these components depends upon the age of the plant; most Citrus species accumulate substantial quantities of flavonoids during organ development. Another study showed that flavonoids content might be affected by the maturity of the fruit, the post-harvesting treatments and the extracting processes [22]. Juice preparation and the processing of fresh Citrus fruits may decrease flavonoids content by 50% due to their water washing methods [23,24], or by elimination of the richest parts of the fruit [25].

In this review, we gather some relevant data for the most abundant flavonoid component in sweet orange (C. sinensis), sour orange (C. aurantium), mandarin orange (C. reticulata), clementine (C. Clementina), lemon (C. limon), and grapefruit (C. Pardisi) juices and peel. It should be noted that Citrus peel exhibit higher contents of flavanones than other parts of the fruit [11]. Depending on their varieties, grapefruit and sour orange are very rich in naringin. Other Citrus species such as sweet orange, and lemon have low quantities of naringin (Table 3). Pupin et al. [26] studied the composition of flavanone glycosides in various orange juice varieties and concluded that narirutin and hesperidin are the most abundant compounds. Hesperidin is present in high levels in clementine, sweet orange, mandarin orange and lemon (39.9–20.5 mg/100 mL juice), while naringin (23 mg/100 mL juice) and narirutin (7.6 mg/100 mL juice), glycosides of naringenin, are especially more abundant in grapefruit (Table 3) [27]. In lemon, eriocitrin is present in high level (16.17 mg/100 mL juice) [26]. Didymin is a typical flavonoid glycoside also known as neoponcirin used in Asian countries as a dietary antioxidant [28]. This flavonoid is mainly present in orange, mandarin and grapefruit (1.89–0.30 mg/100 mL juice) (Table 3).

The most abundant flavanones in Citrus peel are naringin andhesperidin. These flavanones are all glycosylated either by rutinose (6-O-α-l-rhamnosyl-d-glucose) or by neohesperidose (2-O-α-l-rhamnosyl-d-glucose) linked in position 7 [28]. Hesperidin (0.002 to 9.42 mg/g peel dry basis) [29,30] is the main flavanone in all lemon cultivars while levels of diosmin and eriocitrin are the lowest [31]. Mandarin peel is rich in hesperidin (3.95 to 80.90 mg/g peel dry basis) [32,33], narirutin (7.66 to 15.3 mg/g peel dry basis) [22,34], and naringin (0.54 to 0.65 mg/g peel dry basis) [32,33]. Naringin is the most abundant flavonoid in grapefruit and bitter orange peel, conferring a characteristic bitter taste (10.26 to 14.40 mg/g peel dry basis) [29,35].

Citrus peels contain also polymethoxyl flavones, such as sinensetin (0.08 to 0.29 mg/g dry bassis), nobiletin (0.2 to 14.05 mg/g dry basis), tangeretin (0.16 to 7.99 mg/g dry basis) and heptamethoxyflavone [26,36,37,38]. The glycosylated flavones are present in small quantities in Citrus peel, such as diosmin, rhoifoline, isorhoifoline, luteolin. Other flavonoids are present in very small quantities in citrus peel, such as flavonols (quercetin, rutin, myricetin, and kaempferol) [39].

Several studies have shown that the extracts of seeds and leaves of Citrus contain high quantities of phenolic compounds, such as flavonoids [40,41]. Naringin is the most abundant flavonoid in grapefruit seeds (0.2 mg/g seeds) [41]. The flavonoid content in Citrus peel is much higher than in the seeds. They appear in plants and foods mostly as glycosides [42].

Table 3. Flavonoid composition of some Citrus juices [4,43,44].

	Flavanones					Flavones				Total Flavonoids Content
	Hesperidin	Narirutin	Naringin	Didymin	Eriocitrin	Diosmin	6,8-di-C-Glu-Diosmetin	6,8-di-C-Glu-Apigenin	Sinensitin	Total Flavonoids Content
Orange	28.6	5.2	-	1.89	0.31	0.09	0.35	5.72	0.37	18.34
Sour orange	-	-	1.97	-	-	0.15	-	-	-	-
Mandarin orange	24.3	3.92	-	1.44	0.31	-	-	-	1.05	-
Clementine	39.9	4.64	0.08	-	-	1.25	0.2	0.5	-	19.23
Lemon	20.5	-	-	-	16.17	3.12	4.95	1.17	-	-
Grapefruit	0.93	7.60	23.0	0.3	0.41	-	-	-	-	-

Mean values are expressed in mg/100 mL of juice.

4. Citrus Flavonoid Extraction Techniques

Citrus flavonoids were discovered to be omnipresent in practically all portions of Citrus fruits from various species [45]. Extraction is a crucial stage in the analytical process, and its success has a significant impact on the quality of the final results [46]. Flavonoids can only be isolated, detected, and characterized after using the appropriate extraction procedure. Generally, to extract bioactive compounds, several processes can be used, many of which have largely remained constant for hundreds of years. All of these strategies share the same goals: (a) extracting selected bioactive chemicals from complicated plant samples; (b) improving analytical method selectivity and avoiding the presence of interferents that could alter the analysis; and (c) improving bioassay sensitivity by increasing the concentration of targeted compounds before analysis [46,47,48].

4.1. Conventional Extraction Techniques

Various traditional extraction procedures can be used to extract bioactive chemicals from plant sources. The recovery of bioactive chemicals from plant matrices, using common solvents, is referred to as conventional extraction (with or without heat treatment) [49]. The majority of these approaches rely on the extraction power of the various solvents in use, as well as the use of heat and/or mixing. The known conventional procedures for extracting bioactive chemicals from plants are (1) maceration, (2) infusion, (3) decoction, (4) hot continuous extraction (Soxhlet extraction), (5) hydrodistillation, and (6) percolation.

4.2. Non-Conventional Extraction Techniques

Degradation of targeted compounds due to high temperatures and long extraction times in solvents is a major problem encountered in classic extraction techniques. On this basis, finding various extraction strategies to overcome this difficulty becomes a critical step to improve extraction efficiency and/or selectivity. Or, using dedicated aids/energy-intensive intrants, such as microwave-assisted extraction [50], pressurized liquid extraction [51], supercritical fluid extraction [52], ultrasound-assisted extraction, cold plasma-assisted extraction [53], high pressure assisted extraction [54], pulsed electric field assisted extraction [55], and enzyme-assisted extraction [56], is well documented in the scientific literature as an efficient alternative. In general, while studying plant-derived chemicals, the method and solvents utilized for extraction must be carefully adopted [57]. In this context, some of the non-conventional extraction methods are discussed.

4.2.1. Ultrasound Assisted Extraction (UAE)

Ultrasound-assisted extraction is a new technology that is being used to extract natural products that previously took many hours to extract using traditional methods. Initially, it was employed to preserve food, but in the last decade, it has also been utilized to extract beneficial substances (mainly polyphenols). Because of the simplicity of the method, benefits such as reduced extraction time, increased extract yield, and the use of water as a solvent, which reduces the usage of organic solvents, are documented. Therefore, to avoid unwanted reactions generated by the UAE and maximize the extraction yield, the extraction parameters (e.g., extraction duration, solvent system, and, if possible, U.S. frequency) should be tuned prior to developing the extraction process [58]. Londoño-Londoño et al. 2010, conducted the extraction of Citrus peel flavonoids from C. sinensis, C. latifolia, and C. reticulata under optimal ultrasonic conditions of 60 kHz, 40 °C, for 1 h, using methanol as a solvent [59].

4.2.2. Supercritical Fluid Extraction (SFE)

Supercritical extraction is a modern technique that uses gases that have exceeded their critical pressure and temperature, resulting in a fluid with qualities in between a gas and a liquid [60]. Supercritical CO₂ extraction (using CO₂ as a solvent, mainly due to its adaptability, availability, and low cost), is a preferred approach for extracting numerous active compounds. Despite the fact that any gas can be employed as a supercritical fluid [61] because flavonoids are polar molecules, SFE requires the presence of a cosolvent, such as ethanol or methanol [62]. A study was carried out to extract nobiletin and tangeritin from C. depressa var Hayata. The authors tested both methanol and ethanol as solvents. Under the conditions reported in their paper, Lee et al. [36] found that SFE provides a higher amount of flavonoids (+7%) than UAE.

This entry is adapted from the peer-reviewed paper 10.3390/app12010029

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