Antioxidant Activities of Polyphenol Containing Extracts from Citrus

Chapter 24

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Antioxidant Activities of Polyphenol Containing Extracts from Citrus G. K. Jayaprakasha, Κ. N. Chidambara Murthy, and Bhimanagouda S. Patil Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 2119 TAMU, College Station, TX 77843

Several fruits and vegetables that posses antioxidant activity have been reported to be rich in polyphenols and are commercially promoted as functional foods. Citrus fruits contain many bioactive compounds such as phenolics, flavonoids, limonoids, carotenoids, sterols and ascorbic acid. Six different varieties of citrusfruitswere extracted with five solvents. The dried extracts were screened for their radical scavenging activity and antioxidant capacity. The total phenolics have been determined by Folin-Ciocalteu method and the results have been expressed as catechin equivalents. The antioxidant capacity of the extracts is in accordance with the amount of phenolics / lycopene / vitamin C present in each fraction and may provide a good source of antioxidants.

Antioxidants can exercise their protective properties at different stages of the oxidation process and by different mechanisms. There are two main types of antioxidants, namely, "primary" (chain breaking, free radical scavengers) and secondary" or "preventive". "Secondary" antioxidant mechanisms may include deactivation of metals, inhibition of breakdown of lipid hydroperoxides to unwanted volatile products, regeneration of "primary" antioxidants, singlet

264

© 2008 American Chemical Society In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


265 oxygen quenching, etc (7). Compounds possessing such activity are known to possess health benefits in preventing/delaying onset of damage of various biologically significant molecules. This intern helps in prevention or delay in onset of some the diseases like, cardiovascular and carcinoma. The commonly used synthetic antioxidants for the lipid peroxidation are BHT, BHA, TBHQ, propyl gallate and trolox. Among natural compounds ascorbic acid, tocopherol, polyphenols and carotenoids are the major ones (2). Phenolics are known to posses higher antioxidant activity compared to vitamins such as A, C and E, when tested against LDL and VLDL models (3, 4). Lipid peroxidation is a complex chain process involving a variety of radicals. The oxidation is influenced by temperature, light, air, physical and chemical properties of the substrate, and the presence of oxidation catalysts or initiators (4, 5). Plants are rich in phenolic compounds of different origins and functions. Most of them are biologically active as antiviral, antimicrobial, anticarcinogens and atherosclerogenic (2, 6). Phenolic compounds are large, heterogeneous groups of secondary plant metabolites that are widespread in the plant kingdom and they have a wide variety of structures. Flavonoids, tannins and phenolic acids are the main phenolic compounds. It is well known that diets rich in fruits and vegetables are capable of preventing or delaying the onset of certain chronic degenerative diseases of aging, including cardiovascular malfunction and common cancers (5, 7). The antioxidant properties of fruits and spices derived phenolic compounds have been extensively studied by using in vitro chemical systems (7-15). These systems have the advantage of being relatively simple and inexpensive to carry out. However, such in vitro assays are very important before going to in vivo or pre-clinical experiments. On the other hand, their relevance to in vivo health-protective activities is uncertain. It is therefore considered prudent to use more than one antioxidant assay system to measure antioxidant activities, as there may be distinct mechanisms involved, resulting in different outcomes, depending on the method of test (16).

Extraction Efficiency of Phenolics Using Different Solvents Lyophilized fruit powder of blood orange, citron, pummelo, rio red, sour orange and navel orange were extracted with five different solvents. The extracts were concentrated under vacuum, lyophilized and stored at -20 °C until further use. Table 1 depicts the yield from citron, blood orange, rio red, sour orange, pummelo and navel orange using different solvents. Methanol extraction of all the citrusfruitsgave highest yield. However, hexane extract gave minimum yield except citron fruit. In our recent studies, it has been reported that the yield of extractable compounds was highest in methanol extract from pomegranates in comparison with the solvents such as ethyl acetate and water (10).

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

266 Table I. Yield (g/lOOg) of Citrus Fruit Extracts Using Different Solvents* Solvents used for extraction

Citrus varieties


Citron Hexane EtOAc Acetone MeOH MeOH:water (80:20)

1.50 20.01 1.30 40.60 7.31

Blood orange 0.83 7.19 2.76 68.01 20.01

Sour Pummelo Rioorange red 0.82 1.6 1.01 3.4 4.21 10.18 2.01 6.7 5.05 40.4 60.50 47.91 2.73 6.9 12.90

Navel orange 0.64 9.12 2.88 21.04 5.71

* values are mean of triplicate trials

Phenolics have been measured using Folin Cio-calteu method and results have been expressed as catechin equivalents (Table II). Ethyl acetate (EtOAc) extract of rio red, sour orange, pummelo and navel orange gave highest phenolics. Hexane extract did not show any phenolics. Water and acetone extraction gave more phenolics in case of citron and blood orange respectively. On the other hand extraction of phenolic compounds from the fruit is commonly achieved with methanol or aqueous methanol (17, 18). Amount of polyphenols were measured using Folin-ciocalteu method. However, the fact based on their chemical reducing capacity relative to catechin. It has been observed that the phenol antioxidant index is a combined measure of the quality and quantity of antioxidants in vegetables (19). Anagnostopoulou et al. (20) reported EtOAc extract from sweet orange peel has maximum phenolics and which confirms our results. In the present study the responses of the extracts in this assay may be ascribed to the variety and quantity of phenolics found in four extracts from two different varieties of citrus fruits. Generally, extraction with hexane will give non-polar compounds like fatty material or some carotenoids. EtOAc will give medium polar carotenoids and phenolics. The other solvents were used for the extraction of polar compounds like aglycones and glucosides of flavonoids and limonoids depending upon their polarity. It is believed that major components for antioxidant activity in edible plants are carotenoids, vitamin C and polyphenolic compounds. Thus, it is necessary to extract these compounds effectively when antioxidant activities are measured. In this perspective, we chose five different polar solvents to extract all the antioxidants from citrus fruits for the measurement of antioxidant activity. The polarities of antioxidant components in each extract are likely to be different. Type of solvents and polarity may affect the single electron transfer and the hydrogen atom transfer, which are key aspects in the measurements of antioxidant capacity. Hence, the selection of extraction solvent is critical for the complex food samples (27-25).


267 Table II. Phenolics Present in Different Citrus Fruits Extracts in mg g' Solvents used for extraction

Citrus varieties used


Citron EtOAc Acetone MeOH MeOH:water (80:20)

1

1.277 2.964 3.928 7.181

Blood orange 2.96 4.88 0.80 0.20

Rio-red 2.91 0.2 0.54 0.86

Sour orange 2.02 1.62 1.24 1.64

Pummelo 9.7 4.3 1.2 1.8

Navel orange 6.4 2.47 0.52 3.6

Citrus Fruits and Their Radical Scavenging Activity Freeze dried fruit powder of six varieties of citrus fruits was successively extracted with hexane, EtOAc, acetone, MeOH and MeOH:water (80:20) for 8 h each separately. All the extracts were freeze dried and stored at -20 °C until further use. The results of free radical scavenging potentials of citrus fruit extracts from citron, blood orange, rio red, sour orange^ pummelo, navel orange and ascorbic acid at 1000 |ig/mL were tested by DPPH method and the results are depicted in Figure 1. Antioxidants react with DPPH, which is a nitrogencentered radical with a characteristic absorption at 517 nm and convert to 1,1,diphenyl-2-picryl hydrazine, due to its hydrogen donating ability at a very rapid rate (12). The degree of discoloration indicates the scavenging potentials of the antioxidant extracts. MeOH:water (80:20) extract of citron, sour orange and rio red; EtOAc extract of blood orange, pummelo and navel orange exhibited maximum free radical scavenging activity, respectively. On the other hand, hexane extract of citron and pummelo; MeOH extract of blood orange, sour orange, rio red and navel orange showed minimum activity (Figure 1). It is known that free radicals cause autooxidation of unsaturated lipids in food (24). The antioxidant activity of the fractions was attributed to their hydrogen donating ability (25). On the other hand, antioxidants are believed to intercept the free radical chain of oxidation and to donate hydrogen from the phenolic hydroxyl groups, thereby forming stable end product, which does not initiate or propagate further oxidation of lipid (26). The data obtained from this study revealed that, certain fractions isolated from citrus fruits are good free radical scavengers and primary antioxidants that react with DPPH radical, which may be attributed to its proton donating ability. Hsiu-Ling et al. (27) reported the antioxidants in the juice and freeze-dried flesh and peel of red pummelo and their ability to scavenge free radicals and compare them with those in white pummelo juice. The total phenolic content of red pummelo juice extracted by



268

Figure 1. Radical scavenging activity of citrus fruit extracts using DPPH method at 1000 jug/mL

methanol (8.3 mg/ml) was found to be significantly higher than that of white pummelo juice (5.6 mg/ml). The carotenoid content of red pummelo juice was also significantly higher than that in white pummelo juice. The ability of methanol extracts offreeze-driedpeel and flesh from red pummelo to scavenge these radicals was 20-40% that of BHA and vitamin C effects. Fresh red pummelo juice is an excellent source of antioxidant compounds and exhibited great efficiency in scavenging different forms of free radicals including DPPH, superoxide anion and hydrogen peroxide radicals.


269


Antioxidant Capacity of Citrus Extracts The different citrus fruit extracts exhibited various degrees of antioxidant capacity (Figure 2). It is difficult to ascertain an order of antioxidant capacities of different extracts because of the differential responses. Maximum antioxidant capacities were observed in the MeOH:water (80:20) extract of citron, hexane extract of blood orange, MeOH extract of sour orange, EtOAc extract of pummelo, navel orange and rio red. The phosphomolybdenum method is based on the reduction of molybdenum (VI) to molybdenum (V) by the antioxidant compounds and the formation of a green molybdenum (V) complex, which has a maximal absorption at 695 nm. Variations in antioxidant capacity of different extracts may be attributed to differences in their chemical composition such as phenolics, ascorbic acid and carotenoids. Our recent results indicated that certain

Navel orange

Pummelo

Sour orange

Rio red

Blood orange

Citron

0

1000 2000 3000 4000 pmol/g of ascorbic acid equivalents

5000

I Hexane m EtOAc a Acetone • Methanol • MeOH.water (80:20) Figure 2. Antioxidant capacity of citrus fruit extracts at 400 jug/mL



270 citrus limonoids are found to posses moderate antioxidant activity (28, 29). The antioxidant activity shown by the citrus fruit extracts may be due to the presence of flavonoids, carotenoids and ascorbic acid (21). Rehman (30) reported citrus peel extracts caused significant changes free fatty acids, peroxide value, and iodine value of refined corn oil during 6 months of storage at 25 and 45 °C as compared to control. These results confirm the findings of earlier workers, who identified phenolic and flavonoid antioxidative compounds in the non-volatile fraction of methanolic extract of citrus peel (31). Many other researchers found that antioxidant activity in the extract of edible and non-edible plant materials due to the presence of phenolic compounds (2, 10, 32). The findings also revealed that the antioxidant property was observed in orange peel ultra-filtered molasses due to the presence of phenols, including numerous flavanones, flavone glycosides, polymethoxylated flavones, hydroxy cinnamates and other miscellaneous phenolic glycosides and amines (33). Correlation between the content of the total phenolics and radical scavenging activity of the citrus fruit extracts has been reported by several authors (33, 34). Some of the studies reported that there is no correlations between the total phenolic content and the radical scavenging activity (33), but in the present study showed very high correlation coefficient of the total phenolics and radical scavenging activity of all the samples (r = 0.99) except MeOH:water (80:20) extract of citron (21, 22). While there are many methods for the total antioxidant determination, most of the methods have their own limitations (33, 34). It was shown that some antioxidant assay methods give different antioxidant activity trends (35). The DPPH approach seems to be rapid and accurate method for assessing the antioxidant activity of fruit and vegetable extracts. The results are highly reproducible and comparable to other free radical scavenging methods such as ABTS (36). Xu et al. (37) correlated the fifteen varieties of citrus juice components with antioxidant capacity. Phenolic compounds and ascorbic acid were identified as possible antioxidants in orange juice. Phenolic compounds were able to scavenge radicals and to chelate metals (38), while ascorbic acid can play a pro-oxidant role in the presence of transition metals. These compounds can also act as antioxidants because of their ability to trap superoxide anions (39). Depending on the concentrations of phenolic compounds and of transition metals, a complex can be formed that facilitates the redox process (40). Recent literature on citrus fruits and antioxidant activity in different models have been summarized and present in Table III. In general, the phenolic compounds at low concentrations show antioxidant behavior. At higher concentrations, they show pro-oxidant behavior; upon further increasing of their concentration, they again show antioxidant behavior. This always depends on the type (position and number of hydroxyl in the molecule) and the concentration of the phenolic compound, as well on that of the transition metal (48). Thus, different citrus fruit extracts at different concentrations showed different degree of antioxidant activity due to the presence various compounds. 2


271


Table III. Recently Reported Research Findings on Antioxidant Activity of Citrus Fruits Materials employed Pummelo flesh and peels

Activity reported DPPH, superoxide anion, and hydrogen peroxide

Huyou (Citrus paradisi Changshanhuyou) peel 'Cara cara' navel orange (Citrus sinensis L. Osbeck) 'Red Flesh' navel orange pulp

ABTS method, and ferric reducing antioxidant power (FRAP) assay

42

Malondialdehyde , H 0 , Superoxide dismutase (SOD) and catalase

43

SOD, catalase, guaiacol peroxidase, Ascorbate peroxidase, and dehydroascorbate reductase, glutathione reductase

44

Pericarpium Citri Reticulatae of a new Citrus cultivar

DPPH scavenging, hydroxyl radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and reducing power assay

45

Pomaces of Citrus unshiu Edibale part of citron and blood orange

DPPH, Reducing power assay

46

DPPH, phosphomolybdenum method, NBT reduction assay

21

Edibale part of pummelo and navel orange

DPPH, phosphomolybdenum method, ORAC, ABTS, reducing power

22

Edibale part of rio red, sour orange

DPPH, phosphomolybdenum method, NBT reduction assay, reducing power

23

Citrus unshiu, Citrus Total antioxidant capacities (TAC) by ferric reticulata, and Citrus reducing antioxidant power (FRAP) assay. sinensis

47

Juices from fifteen different citrus varieties

38

+

2

2

Ferric reducing antioxidant power, DPPH assay


Ref 41


272 The results obtained in the present study (27-25) demonstrated that the citrus fruit extracts can effectively scavenge various reactive oxygen species or free radicals under in vitro conditions. This may be due to the number of stable oxidized products that can form after oxidation or radical scavenging. The broad range of activity of the extracts suggests that multiple mechanisms are responsible for the antioxidant activity. The multiple antioxidant activity of extracts demonstrated in this study clearly indicatesd the potential application value of the citrus fruits. However, the in vivo safety of extracts needs to be thoroughly investigated in experimental rodent models prior to its possible application as an antioxidant ingredient, either in animal feeds or in human health foods. The above results demonstrated that, some of the extracts could exhibit antioxidant properties, which are comparable to commercial synthetic antioxidants.

Structure Activity Relation and Antioxidant Activity of Citrus Flavanones Antioxidant activity of flavanones depends upon the arrangement of functional groups. The spatial arrangement of substitutents influences the antioxidant activity more than the flavan backbone. The configuration and the total number of hydroxyl groups influence several mechanisms of the antioxidant activity. This is important in flavanones with a substitution of the neohesperidoside group in the 7th position. This may be due to presence of the aglycone form, the 3', 4'-dihydroxy substitution does not influence much on the antioxidant activity. On the other hand, in the flavanones glycosylated with a neohesperidose of the 7 OH group, the 3', 4'-catechol structure the antioxidant power is noticeably increased (49). The difference in antioxidant capacity between the polyhydroxylated and the polymethoxylated flavanones (PMF) is due to the differences either in hydrophobicity or in molecular planarity. The influence of the O-methylation in the aglyconic flavanones is negligible. On the other hand, in case of flavanones such as neohesperidoside molecule has methoxyl group at 7 position and its antioxidant activity has been decreased noticeably as compared to PMF's. Moreover, in the flavanones glycosylated with a neohesperidose of the 7 hydroxyl group, the 3', 4'-catechol structure noticeably increased the antioxidant power and it can be observed in case of neoeriocitrin and naringenin. Oxidation of a flavonoid occurs on the B-ring when the catechol is present yielding a fairly stable ortho-semiquinone radical through facilitating electron derealization. Flavanones glycosylated with neohesperidose lacking catechol system form relatively unstable radicals and are weak scavengers (50). Further, due to the presence of electron-donating groups makes the aromatic system rich in electrons. This confers a higher degree of instability to the flavanone phenoxyl th

th

th


273 th

radicals. Therefore, it could be hypothesized that the sugar molecule in the 7 position is able to interact with the methoxyl group in the 4 position and reduce the antioxidant power (57) th


OH O Naringenin

OH O Heridictyol

CH2OH

OCHj OHOH

01 OH O

HO

OH

Naringin

Hesperidin

OCH, OCH*

CH3O.

OCH> CH3O Neohesperidin

6CH3

O

Tangeretin

Figure. 3. Structures of citrus flavanoids

Flavanone glucosides such as neohesperidin showed an antioxidant power comparable to free flavanones. Hesperitin has shown to have an antioxidant activity higher than the neohesperidin. OGlycosylation at hydroxyl position influences radical-scavenging activities and this could be caused by the steric effect which perturb the planarity and ability to delocalize electrons (57). The antioxidant activity of hesperidine with hesperitin is negligible. Therefore, it could be hypothesized that the kind of sugar in the 7th position (neohesperidose or rutinose) and the position of methoxyl group (3' or 4' position) influence the ability to delocalize electrons. Antioxidants represent a group of substances very important in diet but we cannot ignore that these compounds, when in high concentrations or in particular environmental conditions, can act like prooxidants inducing radicals reaction. In conclusion, further studies are needed on the isolation and characterization of individual compounds from hexane, EtOAc, acetone, MeOH


274 and MeOH:water extracts to elucidate their different antioxidant mechanisms and the existence of possible synergism, if any among the compounds.

Acknowledgements


This project is based upon work supported by the USDA CSREES IFAFS #2001-52102-02294 and USDA-CSREES # 2006-34402-17121 "Designing Foods for Health" through the Vegetable and Fruit Improvement Center.

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Antioxidant Activities of Polyphenol Containing Extracts from Citrus

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