Characterization of Pomegranate's Health ... - ACS Publications

Downloaded by IMPERIAL COLLEGE LONDON on February 18, 2015 | http://pubs.acs.org Publication Date (Web): December 16, 2014 | doi: 10.1021/bk-2014-1185.ch010

Chapter 10

Characterization of Pomegranate’s Health Benefiting Bioactive Compounds, Taste, Color, and Post-Harvest Fruit Quality by Studying a Wide Collection of Diverse Accessions Lior Rubinovich,1 Doron Holland,2 and Rachel Amir*,1 1Migal

Galilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel 2Institute of Plant Sciences, Agricultural Research Organization, Newe Ya’ar Research Center, Ramat Yishay 30095, Israel *E-mail: [email protected].

Pomegranate (Punica granatum L.) is known to be one of the healthiest fruits and its traditional importance as a medicinal plant is supported by modern science. The health beneficial properties of the fruit are attributed to its high levels of antioxidant compounds, mainly hydrolysable tannins (HTs) and anthocyanins. This review focuses on our recent analyses of a wide, bio-diverse pomegranate collection composed of 29 different accessions. Our aim was to compose a wide-scope picture of the various factors that contribute to the health benefits, and to the marketability of the fruit, in particular antioxidant compounds, taste and color. For that purpose we have examined the concentration and localization of HTs, anthocyanins, total phenols, organic acids and total soluble sugars (TSS) in the fruit sections. We have also examined how these factors are affected by environmental conditions. In addition, we have investigated the factors that help maintain fruit quality during prolonged storage and how they may be utilized to control storage diseases and reduce the use of synthetic fungicides. The usage of a large pomegranate collection rich in trait variation is a valuable and powerful resource to increase our knowledge on the biodiversity that can be found in pomegranates. It can also give us insights on © 2014 American Chemical Society In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

pomegranate biology and help us to determine which bioactive compounds and factors regulate the fruits taste. Besides the obvious benefits to basic science, the overall collected data can assist breeders and growers to respond to consumer and industrial preferences.


Introduction Fruits and vegetables contain high levels of antioxidant compounds that protect against harmful free radicals. The antioxidant activity is attributed mainly to high total phenols content (TPC). Phenols, in addition to their ability to serve as scavengers of free radicals and reactive oxygen species (ROS), have been associated with the reduction of stress-related chronic diseases and age-related disorders, such as cardiovascular diseases, carcinogenesis, neurodegeneration, skin deterioration as well as other health benefits (1). Pomegranates (Punica granatum L.) have high TPC (2, 3) and strong antioxidant activities (4–6). It was shown that pomegranate juice (PJ) possess a 3-fold higher antioxidant activity than that of red wine or green tea (3), and two-, six- and eight-fold higher levels than those detected in grape/cranberry, grapefruit, and orange juice, respectively (6, 7). Moreover, the pomegranate fruit is known to be one of the healthiest fruits and its traditional importance as a medicinal plant (8) is now supported by data obtained from modern science showing that the fruit contains anti-carcinogenic (9, 10), anti-microbial (11), antifungal (12) and anti-viral compounds (13) as well as many other health beneficial activities. Recent biological studies have also proven that certain compounds contained in PJ reduce blood pressure, are anti-atherosclerotic and significantly reduce low density lipoprotein (LDL) oxidation (2, 4, 5, 14, 15). Chemical analyses have shown that the phenol fraction of PJ contains a high level of hydrolysable tannins (HTs) as well as anthocyanins, which exhibit high antioxidant activities (3, 16). Anthocyanins are water-soluble pigments primarily responsible for the attractive red-purple color of many fruits, including PJ, and are well known for their antioxidant activity (17). An analysis of PJ prepared by hydrostatic pressure applied to the whole fruit showed that the predominant type of phenolic compounds extracted from the peels during the process are water-soluble HTs; these compounds account for 92% of the fruit antioxidant activity (3). HTs are found in the peel (husk, rind, or pericarp), carpellary membranes , and piths of the fruit (18). The main compounds in this group are the punicalagin isomers, which were suggested as being responsible for about half of the total antioxidant capacity of the juice. In addition, ellagic acid, gallagic acid, and punicalin were also suggested to play a significant role in this acativity (3). The goal of this review is to demonstrate how studies conducted with a large collection of diverse accessions, can help to elucidate different factors that contribute to the health benefits and the marketing of the fruit. Collections of wild and domesticated pomegranates accessions are available in Asia, Europe, North Africa, and North America (19–21). This review is mainly based on our 202 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


recent studies performed on 29 pomegranate accessions that were chosen out of several hundred accessions from a collection in the Newe Ya’ar research center, ARO [registered in the Israel Gene Bank for Agriculture Crops (IBG, Website: http://igb.agri.gov.il)] (22) (Figure 1) that differ in their size, taste, color, aril hardness and ripening date. This review concentrates on: (i) Measurement and elucidation of the bioactive compounds, and defining their localization in the fruits sections; (ii) Analysis of the effect of two distinct growing habitats on pomegranate health-promoting components; (iii) Elucidation of factors that influence the pomegranate taste; (iv) assessment of the parameters affecting pomegranate color and (v) Studying factors that keep the fruit and especially the peel quality during prolonged storage conditions.

Figure 1. The 29 pomegranate accessions used in this study. Reprinted with permission from Tzulker, R.; Glazer, I.; Bar-Ilan, I.; Holland, D.; Aviram, M.; Amir, R. J. Agric. Food Chem. 2007, 55, 9559–9570. Copyright 2007 American Chemical Society. (see color insert) 203 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

204


Table 1. Correlation Matrix between Antioxidant Activities Measured by Different Methods, Total Polyphenols, Total Anthocyanins and the Level of the Four Hydrolysable Tannins in Juice Prepared from the Arils of the 29 Pomegranate Accessions According to the Pearson Test.

Antioxidant Activity FRAP Antioxidant Activity DPPH Total Polyphenols Total Anthocyanins Punicalagin

Antioxidant Activity FRAP

Antioxidant Activity DPPH

Total Polyphenols

Total Anthocyanins

Punicalagin

Punicalin

Gallagic Acid

1

0.83**

0.86**

0.68**

0.16

-0.02

0.1

1

0.62**

0.265

0.48**

0.12

0.39*

1

0.71**

-0.1

0.01

-0.06

1

-0.34

-0.14

-0.31

1

0.14

0.45*

1

0.79**

Punicalin

1

Gallagic Acid

The r value of the correlation is given and its significance (p < 0.05) is identified by one asterisk, while (p < 0.01) is identified by two asterisks. The table is modified from Tzulker et al., 2007.

In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

205


Table 2. Correlation Matrix (Pearson Test) Conducted on the Data Obtained from Homogenates Prepared from the Peels Alone of 29 Pomegranate Accessions. The r Value of the Correlation Is Given and Its Significance (p < 0.05) Is Identified by One Asterisk, while (p < 0.01) Is Identified by Two Asterisks.

Antioxidant Activity FRAP Antioxidant Activity DPPH Total Polyphenols Anthocyanins Punicalagin Ellagic Acid

Antioxidant Activity FRAP

Antioxidant Activity DPPH

Total Polyphenols

Total Anthocyanins

Punicalagin

Ellagic Acid

Punicalin

Gallagic Acid

1

0.51**

0.95**

0.29

0.63**

0.70**

0.85**

0.94**

1

0.55**

0.11

0.29

0.33

0.31

0.43**

1

0.28

0.63**

0.77**

0.87**

0.93**

1

0.07

0.41*

0.27

0.41*

1

0.27

0.6**

0.68**

1

0.70**

0.72**

1

0.85**

Punicalin

1

Gallagic Acid

aThe r value of the correlation is given and its significance (p < 0.05) is identified by one asterisk, while (p < 0.01) is identified by two asterisks. The table is modified from Tzulker et al., 2007.

In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Elucidation of the Bioactive Compounds That Contribute to the Antioxidant Activity and Their Localization in the Fruits Sections In order to define the major bioactive compounds that contribute to the antioxidant activity, and to reveal where they are localized in the fruits sections (peels, arils, seeds, carpellary membranes), we have used 29 unique accessions (23). Arils and peels were separated from the fruits, arils juice was prepared by squeezing the arils and separating them from their seeds, while the peels were homogenized with water. We also obtained juice which contained the aril juices with compounds extracted from the inner peels using a fruit juice extractor. The results demonstrate that in arils juice, the range of antioxidant activity is about 3 fold and the range of TPC between accessions is about 2.5 fold from the lowest to the highest (23, 24). However, the range of the total anthocyanins contents was about 33-fold difference between the accessions. The antioxidant activity in aril juice correlated significantly to TPC and to the total anthocyanin contents, but not to the levels of four members of HTs (punicalagin, punicalin, gallagic and ellagic acids, Table 1) (23, 25). Anthocyanins are well known to contribute to the antioxidant activity (17). Therefore, when consuming only the arils, the accessions with the highest health benefiting properties are those that have red or darker colored arils, since these have higher total anthocyanins content. It is worth mentioning that the correlation value between the total anthocyanin content and antioxidant activity was significant, but quite low (r=0.68). This implies that anthocyanins are only one of the contributors to the antioxidant activity, and that other yet unknown compounds also contribute to this activity. Measurements of the antioxidant activities of juice prepared by juice extractor and of peels homogenates, have shown that the activity levels were approximately 5-fold and 40-fold higher than that measured in aril juice, respectively (23). This demonstrates that the peels contain compounds that have high antioxidant activity, as previously suggested (3, 26). We have also found that in peels, the ranges of antioxidant activity and TPC within the 29 accessions were about 5 fold and 3 fold respectively. To assess the levels of HTs that were previously suggested to contribute to the antioxidant activity (17), the contents of four members of this group (punicalagin, punicalin, gallagic and ellagic acids), were measured in the peels homogenates (23). Their levels were significantly correlated to the antioxidant activity (r= 0.63, r= 0.85, respectively), suggesting that they are major contributors to this activity in the peels homogenates (Table 2). The levels of punicalagins isomers were about 5 x 103 fold higher in the peels compared to the arils. No correlation was found between the level of anthocyanins to the antioxidant activities of the peels homogenates (23). All in all, the results of this study have shown that juice prepared from the arils alone exhibit relatively poor antioxidant activity and low TPC, as well as low content of the four HTs, relative to homogenates prepared from the peels. Therefore, in order to achieve maximal health benefits from pomegranate consumption, PJ should be prepared with the proper means which can also extract 206 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


the peels along with the arils, since peels contain significantly higher levels of compounds that contribute to the antioxidant activity and human health. A recent study that was carried out on Wonderful accession, has shown that the inner peels have higher levels of antioxidant capacity and higher levels of the examined HTs than the external peels (27). Thus, future studies may be carried out to determine to which level the peels should be extracted along with the arils and to examine the effect of this process on the sensory properties of PJ. Moreover, it would be beneficial to inspect the range and nature of variation in the above mentioned properties of already commercialized pomegranate products in order to elevate its quality.

The Effect of the Habitat on Health-Promoting Compounds Due to the extensive knowledge about the pomegranate’s health attributes noted above and increasing public awareness of functional foods, the demand for pomegranate fruit and its by-products has increased tremendously in the Western world. As a result, the land area devoted to pomegranate orchards has increased significantly, including plantations in different geographic regions having diverse growth condition. In order to gain more knowledge of how environmental conditions affect the antioxidant activity and the levels of HTs, we compared the properties of pomegranates grown in two different habitats for two consecutive years. The habitats compared were a Mediterranean temperate and a hot dry desert climate (24). Eleven out of the 29 accessions were chosen for the study (Figure 2). Our findings revealed that in most of the accessions, the aril juice obtained from fruits grown in Mediterranean temperate had a higher antioxidant activity than those grown in hot and dry desert climate, with some exceptions. Consistent with these findings, it was found that the level of total anthocyanins was significantly higher in aril juice of fruits obtained from Mediterranean climate compared to those from desert climate (up to 40-fold). Anthocyanins level highly correlated to the antioxidant levels and to TPC in Mediterranean climate (r= 0.84; r = 0.70, respectively), but less in desert climate (r=0.34; r=0.61, respectively). Temperature is an important factor that affects anthocyanin accumulation in plants and it was shown that the expression of anthocyanin biosynthetic genes has been induced by low temperature and repressed by high temperature in various plants (28–33). The higher level of anthocyanins in Mediterranean climate fruits could be attributed to the relatively lower temperatures in this habitat compared to desert climate. In contrast, examination of juices obtained using a juice extractor, which include in addition to the aril juice the extract of the inner parts of the peels, as well as peel homogenates, show that antioxidant activity and TPC were significantly higher in fruits obtained from desert climate (up to 4-fold). The antioxidant capacity in the peels significantly correlated to the levels of punicalagin and punicalin in both habitats (Mediterranean climate: r=0.33, r=0.68; desert climate: r=0.64, r=0.34 respectively). The reasons for the higher TPC in the peels of pomegranates grown in desert climate are not clear but could 207 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


be attributed to the higher temperatures and radiation found in desert climate compared to Mediterranean climate. The higher phenolic compounds content might protect the fruits and seeds against oxidative stress occurring under the prevailing climatic conditions. In summary, the study of eleven accessions indicates that environmental conditions significantly affect the concentration of pomegranate fruit health beneficial compounds. In addition, the high content of health-promoting components in the peels of fruits grown in a desert climate may be advantageous to the byproduct nutraceuticals industry. A comprehensive study consisting a wider diversity of germplasm may be carried out in order to construct a more complete picture on the effect of environmental conditions on pomegranate fruits parameters.

Figure 2. Fruits of the 11 pomegranate accessions grown in two different habitats as photographed in the 2006 and 2007 seasons. The figure is modified from Schwartz et al., 2009. (see color insert)

Parameters Affecting Pomegranate Taste Analysis of a large collection can also shed more light on the compounds that are responsible and contribute to the taste of PJs. As for many fruit species, pomegranate varieties differ in their taste, ranging from sweet to sour (22). This is related directly to the quality and quantity of the organic acids and sugars found in the fruit, and indeed a great diversity in these components and their contents 208 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


has been detected in different pomegranate juices collected from several regions around the world (34–37). The desired pomegranate taste varies, however, in different countries and regions. In North Africa, for example, almost all commercialized pomegranate belong to the sweet varieties (38), while in Russia and other northern countries more sour accessions are commercialized (39). Total soluble solids (TSS) and titratable acidity (TA) are two parameters who’s ratio define the taste of PJ. Determination of TSS is important not only to establish the organoleptic quality of the juice, but also because TSS content is the major parameter determining the accessions that can be used for wine preparation. Acidity can play an important role in the perception of fruit quality. It not only affects the fruit’s sour taste but also its sweetness by masking the taste of the sugars. Thus, ratio between TSS and TA is commonly used to define the “taste” of a fruit, and overall consumer appreciation is related more to TSS/TA ratio than to soluble sugars content alone (40). To determine the compounds that contribute to the taste of the aril juices, the levels of TSS and TA were measured in arils juice of the 29 accessions (41). The results showed that in arils juice: (i) There is no significant difference in the TSS and the sugars among the 29 accessions (15 to 18.5% TSS), implying that they are not the main contributors to taste; (ii) The levels of TA and citric acid do differ significantly among these accessions (by 15-fold and 25-fold, respectively), suggesting that they play a major role in determining juice taste; and (iii) Citric acid is predominant in the sour accessions, while oxalic and succinic acids are major in sweet accessions. These results suggest that since the level of sugars and TSS do not alter significantly between the different accessions, TA and citric acid are the main contributors to the taste, which as mentioned above, is determined by the ratio between TSS and TA. This ratio ranged significantly (from 6.1 to 64.6) between the different accessions. We have also studied the levels of these taste parameters in the peels of the different accessions. Although pomegranate peels are inedible due to their bitter taste and tough dry texture, some pomegranate juice industries squeeze the fruits in such a way that in addition to the arils, the compounds found in the peels are also extracted (3). Therefore, it is important to study the contents of sugars and organic acids in the peels whose ratio defines the taste of PJ. The peel homogenates exhibited lower levels of TSS compared to the aril juice (by about 2-3 folds). In addition to glucose and fructose, which were also found in arils juice, the homogenates contained also maltose (whose concentration differs among the accessions by about 50-fold), sucrose that was found in only six accessions, and mannitol whose concentration differs between the accessions by about 8-fold. The levels of TA were within the range found in aril juices and the concentration range of TA and citric acid within the different accessions was about 3 and 30 fold, respectively. Oxalic acid was detected only in a few accessions. Exploring the relationship of TA and TSS values between the arils and the peels could lead to better understanding of the factors regulating pomegranate juice qualities, and to gain more information about the regulation of these parameters. For that, we have performed a correlation matrix and found that the levels of TA and citric acid are significantly positively correlated, but no correlations were found in the levels of TSS and soluble sugars (Table 3). 209 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

210


Table 3. Correlation Matrix (Spearman Test) Conducted on Data Obtained from the Peels and Aril Juices of 29 Accessions Peels Arils

TA

TSS

citric acid

glucose

fructose

Anthocyanin

TA

0.62**

-0.21

0.78**

-0.1

-0.08

-0.28

TSS

0.75**

0.22

0.61**

-0.08

-0.08

0.36

citric acid

0.67**

-0.33

0.88**

-0.19

-0.15

-0.36

glucose

0.53**

0.26

0.32

-0.15

-0.16

0.37*

fructose

0.52**

0.26

0.31

-0.15

-0.15

0.37*

Anthocyanin

0.41**

0.07

0.39*

0.24

0.17

0.26

The r value of the correlation is given and is significance (p

Characterization of Pomegranate's Health ... - ACS Publications

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