CHAPTER 2

REVIEW OF LITERATURE

To justify the significance of this research on ellagitannin extraction from pomegranate

peel, storage stability and application of ellagitannin, it was necessary to obtain detailed

information from the available literature. The relevant information from the literature has

been collected and critically reviewed under the following sections:

• Pomegranate fruits, variety, production and its processing

• Pomegranate fruit parts and composition

• Pomegranate phytochemicals, Ellagitannin’s chemistry and biochemistry

• Ellagitannin (Polyphenols) extraction techniques

• Extraction process optimization and statistical calculation methodology

• Studies on isolation and purification of ellagitannins extract

• Physicochemical and functional characterization studies of ellagitannin

• Storage stability of ellagitannin

• Application of ellagitannin

2.1 Pomegranate fruit, variety, production and its processing

2.1.1 Pomegranate Fruit

The pomegranate (Punica granatum L.) is an ancient fruit and many facts are available

in history (Damania, 2005). It is being considered that pomegranate fruit is one of the

9

first 5 crops along with Olives, Figs, Grapes and Dates, to be cultivated. Domestication

of pomegranate started from 3000-4000 BC in Iran, Afghanistan and Turkey from

where it travelled to other part of world e.g. Mediterranean countries, India, China.

(Arjmand, 2011). In many culture and religion pomegranate fruit is considered as holy

fruit. In Greek mythology it represents life, regeneration and marriage, in Judaism 613

number of pomegranate seeds are linking with Bible’s 613 commandments, in

Buddhaism it represents the essence of favorable influences, in China as a ceramic art

symbolizing fertility, in Christianity as a symbol of resurrection and eternal life and in

Islam the Quran describes four gardens with springs, shade and fruits including the

pomegranate. The pomegranate plant is being used as juice, dyes inks, tannins for

leather and giving remedies in different ailments. The pomegranate fruits having a long

shelf life at room temperature, due to which it has carried on long journeys and highly

utilized in desert climates as a source of water and nourishment (Langley, 2000).

2.1.2 Botanical background of pomegranate

Pomegranate fruit botanically classified as berry. Pomegranate grows as a small tree

reaching 4-10 m. The fruit size can vary from 6-12 cm in diameter and has a tough,

leathery skin. The pomegranate fruit developed from an inferior ovary and synchronous

pistil with two whorls of basal carpels lying within the receptacle. During the

development of ovary, the outer carpels become tilted up and superimposed (Still,

2006).

10

2.1.3 Taxonomy of pomegranate

Engler and Prantl’s (1931) Hutchinson (1959)

Division : Angiospermae Phylum : Angiospermae

Class : Dicotyledonae Subphylum : Dicotyledonae

Subclass : Archichlamydeae Division : Lignosae

Order : Myrtiflorae Order : Myrtales

Table 2.1 Vernacular names of pomegranate (Morton, 1987)

Indian Language

Vernacular Name

International Language

Vernacular Name

Bengali Dalim Chinese Shiliu Gujrati Dadam French Grenade Hindi Anar German Granatapfel Kannada Dalimbe Hebrew Rimon Malayalam Matalam Italian Melogranate Oriya Dalimba Japanese Zakuro Punjabi Anar Romanian Rodie Sanskrit Darimba Russian Granatnik Tamil Madulam Spanish Granada Telugu Dhanimmapandu Swedish Granatäpple

2.1.4 Pomegranate Variety in India

Major pomegranate varieties grown in India are listed in Table 2.2. Among all varieties

grown in India, variety of Bhagwa is easily available on commercial scale and is a

medium size fruits having soft seeds, redish flesh and sweet juice.

11

Table 2.2 Indian verities of pomegranates and their characteristic features

S. No. variety name of Pomegranate Characteristic features

1 Ganesh

This variety is developed by selection method. It is a prolific bearer, fruit very large, rind yellowish red, pinkish aril with soft seeds. It is the commercial cultivar of Maharashtra. The average yield ranges from 8-10 kg per tree

2 Bhagwa The fruit is glossy red in colour with soft seeds and high T.S.S. commercial variety in Maharashtra

3 Arakta The fruits are smaller than Ganesh variety having dark red coloured arils with soft seeds.

4 Mrudula

This variety has all the characters of the Ganesh variety except the arils are dark red in colour. The colour of the arils in 'Ambe' bahar and 'Mrig' bahar is dark red in colour while it is pink during the 'Hasta' bahar. The average fruit weight is 250-300 grams

5 Muskat The fruits of this variety have red rind with pink coloured arils. The fruit are with average weight of 300-350 grams

6 Jyoti

This variety was developed at IIHR, Bangalore. The fruits are large with attractive colour having dark red arils. The seeds are very soft with high pulp and juice contents. Fruits are borne on the inner side of the canopy and thus do not get damaged due to sun scorching

7 Ruby

This variety is developed at IIHR, Bangalore. The mature fruits resemble cultivar 'Ganesh' with respect to shape and size. However, the rind of this variety is reddish brown with green streaks containing red bold arils. The fruit weighs 270 g with an average yield of 16-18 tonnes/ha.

8 Dholka Fruits large, rind yellowish red with pinkish white aril. It is a popular cultivar of Gujarat

9 Kandhari Fruit large in size, rind deep red, fleshy testa, blood red or deep pink with sweet, slightly acidic juice, seeds hard

10 Sindhoor Prolific yield, medium to large size fruits, soft seeds, red flesh and sweet juice

11 Bedana Fruit medium in size, rind brownish or whitish: fleshy testa pinkish white with sweet juice; seeds soft

(Source: National Horticulture Board, India, 2014)

12

2.1.5 Pomegranate production

According to the data published by National Horticulture Board of India, the

pomegranate cultivation area in India is 131 thousand ha and production is around 1346

thousand tons in the year of 2013-2014. Maharashtra is the leading producer of

pomegranate followed by Karnataka, Andhra Pradesh, Gujarat and Tamil Nadu. In India,

pomegranate is commercially cultivated in Solapur, Sangli, Nasik, Ahmednagar, Pune,

Dhule, Aurangabad, Satara, Osmanabad and Latur districts of Maharashtra; Bijapur,

Belgaum and Bagalkot districts of Karnataka and to a smaller extent in Gujarat, Andhra

Pradesh and Tamil Nadu.

Table 2.3 Area and Production of Pomegranate in India

Year Area (HA) Production (MT) 2008-09 109000 807000

2009-10 125000 820000

2010-11 107000 743000

2012-13 125000 820300

2013-14 131000 1346000

(Source: National Horticulture Board of India)

Table 2.4 Average Price of pomegranate in India year wise

Year Average Price (Rs. per Quintal)

2014 8890

2013 8446

2012 8156

(Source: National Horticulture Board of India, 2014)

13

Table 2.5 Pomegranate storage condition

Temperature (°C) 5 - 7

Relative Humidity (%) 90 - 95

Storage Period (months) 2 - 3

(Source: National Horticulture Board of India, 2014)

2.2 Pomegranate fruit parts and composition

Pomegranate fruit parts has reviewed as below:

2.2.1 Structure and parts of pomegranate fruit The fruit’s internal structure is irregular many segments having many closely packed red

seeds (arils). Each irregular segments separated by non-edible white piths and thin

carpellary membranes. The pericarp (peel) of fruit is tough and leathery, and seeds (arils)

are anchored on a soft and fleshy placental (pith) tissue. Each aril contains a seed

surrounded by edible acidic juicy pulp (Figure 2.1). The USDA has established the

nutritional facts and composition of different parts of pomegranate fruits. Composition of

pomegranate fruit and their edible part are shown in Figure 2.2, Table 2.6 and Table 2.7

14

Figure 2.1 Pomegranate whole fruit, internal view and its parts

15

2.2.2 Composition of pomegranate fruit

Figure 2.2 Schematic presentation of pomegranate fruit part and their composition

16

Table 2.6 Nutritional composition of pomegranate edible part (USDA, 2007)

Nutrients Value

Water (g/100g) 80.97

Energy (Kcal/100g) 68

Protein (g/100g) 0.95

Fat (g/100g) 0.30

Carbohydrate (g/100g) 17.17

Dietary Fiber (g/100g) 0.6

Total sugar (g/100g) 16.57

Vitamin C (mg/100g) 6.1

Vitamin A (IU/100g) 108

Vitamin E (α-tocopherol) (mg/100g) 0.60

Vitamin K (Phyloquinones) (µg /100g) 4.6

Phytosterols (mg/100g) 17

Cholesterol (mg/100g) 0

α –carotene (µg /100g) 50

β-carotene (µg /100g) 40

Table 2.7 Mineral content of the edible part (USDA, 2007)

Minerals Values (mg/kg) Calcium 30 Magnesium 30 Potassium 2590 Sodium 30 Iron 3.0 Copper 0.7 Zinc 1.2

17

2.3 Pomegranate phytochemicals

The phytochemicals that have been identified from various parts of the pomegranate tree,

fruits and seeds are listed in Table 2.8, the major class of pomegranate phytochemicals is

the polyphenols (phenolic rings with multiple hydroxyl groups) that is abundant in the fruit.

Pomegranate polyphenols include flavonoids (flavonols and anthocyanins), condensed

tannins (proanthocyanidins) and hydrolysable tannins (ellagitannins and gallotannins)

(Figure 2.3). Other phytochemicals identified from the pomegranate are organic and

phenolic acids, sterols and triterpenoids, fatty acids, triglycerides, and alkaloids (Seeram et

al, 2006).

The world market for polyphenols is significant. For example, Leather head Food

Research (2009) has estimated the market of polyphenols of worth approximately $200

million. The majority of polyphenols extracted for sale as nutraceuticals or for use in

functional foods come from grapes, apples, olives and green tea.

The flavonoids include flavonols such as luteolin, quercetin, and kaempferol found in the

peel extract (Elswijk et al, 2004) and anthocyanins found in the arils. Anthocyanins are

water soluble pigments responsible for the bright red color of PJ (Hernandez et al, 1999;

Santagati et al, 1984). Hydrolyzable tannins are available in the peels (rind, husk, or

pericarp), membranes, and piths of the fruit (Seeram et al, 2005)

18

Figure 2.3 Classification of tannin (Khanbabaee and van Ree, 2001)

Table 2.8 Pomegranate phytochemicals (Seeram et al, 2006a)

S. No.

Name of phytochemical Chemical Formula

Molecular weight

Plant part

Ellagitannins and Gallotannins

1

2,3-(S)-HHDP-D-glucose C20H18O14 482.35 Bark, peel

2 Castalagin C41H26O26 934.63 Bark 3 Casuariin C34H24O22 784.54 Bark

4 Casuarinin C41H28O26 936.65 Bark,

pericarp

5 Corilagin C27H22O18 634.45 Fruit,

leaves, pericarp

19

6

Cyclic 2,4:3,6-bis(4,4’,5,5’,6,6’- hexahydroxy[1,1’-

biphenyl]- 2,2’-dicarboxylate) 1-(3,4,5-trihy droxybenzoate) b-

D-Glucose

C41H28O26 936.65 Leaves

7 Granatin A C34H24O23 800.54 Pericarp 8 Granatin B C34H28O27 952.64 Peel

9 Pedunculagin C34H24O22 784.52 Bark,

pericarp 10 Punicacortein A C27H22O18 634.45 Bark 11 Punicacortein B C27H22O18 634.45 Bark 12 Punicafolin C41H30O26 938.66 Leaves 13 Punigluconin C34H26O23 802.56 Bark 14 Strictinin C27H22O18 634.45 Leaves

15 Tellimagrandin I C34H26O22 786.56 Leaves, pericarp

16 Tercatain C34H26O22 786.56 Leaves

17 2-O-galloyl-4,6(S,S) gallagoyl- D-glucose

C41H26O26 934.63 Bark

18 5-O-galloyl-punicacortein

D C54H34O34 1222.8 Leaves

19 Punicacortein C C47H26O30 1070.7 Bark

20 Punicacortein D C47H26O30 1070.7 Bark,

heartwood

21 Punicalin C34H22O22 782.53 Bark,

pericarp

22 Punicalagin C48H28O30 1084.7 Bark,

pericarp, peel

23 Terminalin/gallayldilacton C28H20O16 602.37 Pericarp Ellagic Acid Derivatives

24 Ellagic acid C14H6O8 301.19 Fruit,

pericarp, bark

25 Ellagic acid, 3,3’-di-O-

methyl C16H10O8 330.25 Seed

26 Ellagic acid, 3,3’, 4’-tri-O- Methyl

C17H12O8 344.27 Seed

27 Ellagic acid, 3’-O-

methyl-3, 4-C16H8O8 328.23 Heartwood

20

methylene 28 Eschweilenol C C20H16O12 448.33 Heartwood

29 Diellagic acid rhamnosyl(1-4)

glucoside C40H30O24 894.65 Heartwood

Catechin and Procyanidins 30 (-)-Catechin C15H14O6 290.27 Juice

31 Catechin-(4,8)-gallocatechin

C30H26O13 594.52 Peel

32 Gallocatechin C15H14O7 306.27 Peel

33 Gallocatechin-(4,8)-

catechin C30H26O13 594.52 Peel

34 Gallocatechin-(4,8)- gallocatechin

C30H26O14 610.52 Peel

35 Procyanidin B1 C30H26O12 578.52 Juice 36 Procyanidin B2 C30H26O12 578.52 Juice

Anthocyanins and Anthocyanidins 37 Cyanidin C15H11O6 287.24 Juice 38 Cyanidin-3-glucoside C21H21O11 449.38 Juice 39 Cyanidin-3,5-diglucoside C27H31O16 611.52 Juice 40 Cyanidin-3-rutinoside C27H31O15 595.53 Juice 41 Delphinidin C15H11O7 303.24 Juice 42 Delphinidin-3-glucoside C21H21O12 465.38 Juice

43 Delphinidin 3, 5-diglucoside

C27H31O17 627.52 Juice

44 Pelargonidin 3-glucoside C21H21O10 433.38 Juice

45 Pelargonidin 3,5-

diglucoside C27H31O15 595.53 Juice

Flavonols

46 Apigenin-4’-O-β-D-

glucoside C21H20O11 448.32 Leaves

47 Kaempferol C15H10O6 286.24 Peel, fruit 48 Luteolin C15H10O6 286.24 Peel, fruit

49 Luteolin-3’-O-β-D-

glucoside C21H20O10 432.11 Leaves

50 Luteolin-4’-O-β-D-glucoside

C21H20O10 432.11 Leaves

51 Luteolin-3’-O-β-D-

Xyloside C20H18O10 418.09 Leaves

52 Myricetin C15H10O8 318.04 Fruit 53 Quercetin C15H10O7 302.04 Peel, fruit

21

54 Quercimeritrin C21H20O12 464.38 Fruit 55 Quercetin-3-O-rutinoside C27H30O16 610.52 Fruit

56

Quercetin-3,4’-dimethyl ether 7-

O-α-L-arabinofuranosyl-(1-6)- β-D-glucoside

C28H32O16 624.54 Bark, peel

57

Eriodictyol-7-O-α-L- arabinofuranosyl (1-6)-

β-D- glucoside

C26H30O15 582.51 Leaves

58

Naringenin 4’-methylether 7-O-α-L-arabinofuranosyl

(1-6)-β-D-glucoside

C27H32O14 580.53 Leaves

Organic acid 59 Caffeic acid C9H8O4 180.16 Juice 60 Chlorogenic acid C16H18O9 345.31 Juice

61 Cinnamic acid C9H8O2 148.16 Juice 62 Citric acid C6H8O7 192.12 Juice 63 o-Coumaric acid C9H8O3 164.16 Juice 64 p-Coumaric acid C9H8O3 164.16 Juice 65 Ferulic acid C10H10O4 194.18 Juice 66 Gallic acid C7H6O5 169 Juice 67 L-Malic acid C4H6O5 134.09 Juice 68 Oxalic acid C2H2O4 90.03 Juice 69 Protocatechuic acid C7H6O4 154.12 Juice 70 Quinic acid C7H12O6 192.17 Juice 71 Succinic acid C4H6O4 118.09 Juice 72 Tartaric acid C4H6O6 150.09 Juice

Simple Galloyl Derivatives 73 Brevifolin C12H8O6 248.19 Leaves 74 Brevifolin carboxylic acid C13H8O8 292.2 Leaves

75 Brevifolin carboxylic acid-10-monosulphate

C13H7KO10S 394.25 Leaves

76 1,2,3-Tri-O-galloyl-β-D-

glucose C27H24O18 636.47 Leaves

77 1,2,4-Tri-O-galloyl- β -D-

glucose C27H24O18 636.47 Leaves

78 1,2,6-Tri-O-galloyl- β -D-glucose

C27H24O18 636.47 Leaves

22

79 1,4,6-Tri-O-galloyl- β -D-

glucose C27H24O18 636.47 Leaves

80 1,3,4-Tri-O-galloyl- β -D-glucose

C27H24O18 636.47 Leaves

81 1,2, 4, 6-Tetra-O-galloyl-

β -D-glucose C34H28O22 788.57 Leaves

82 1,2,3,4, 6-Pent-O-galloyl-

β -D-glucose C41H32O26 940.68 Leaves

83 Methyl gallate C8H8O5 184.15 Heartwood

84 3,4,8,9,10-pentahydroxy- dibenzo[b,d]pyran-6-one

C13H8O7 276.20 Leaves

Alkaloids 85 Hygrine C8H15NO 141.21 Root bark 86 Norhygrine C7H13NO 127.18 Root bark 87 Pelletierine C8H15NO 141.21 Bark 88 N-methyl pelletierine C9H17NO 155.24 Bark 89 Sedridine C8H17NO 143.23 Bark 90 Pseudopelletierine C9H15NO 153.22 Bark 91 Nor-pseudopelletierine C8H13NO 139.19 Bark

92 2,3,4,5-tetrahydro-6-propenyl-pyridine

C8H13N 123.20 Bark

93 3,4,5,6-tetrahydro-a-

methyl-2-pyridine ethanol C8H15NO 141.21 Bark

94 1-(2,5-dyihydroxy-phenyl)- pyridium chloride

C11H10ClNO2 223.66 Leaves

Other compound

95

Coniferyl 9-O-[ β-D- apiofuranosyl-(1-6)]-O- β-

D-glucopyranoside

C21H30O12 474.46 Seed

96 Sinapyl 9-O-[ β-D-

apiofuranosyl-(1-6)]-O- β-D- glucopyranoside

C22H32O13 504.48 Seed

97 Phenylethyl rutinoside C20H30O10 430.45 Seed 98 Icariside D1 C19H28O10 416.42 Seed 99 Mannitol C6H14O6 182.17 Bark

In commercial pomegranate juice which is obtained by hydrostatic pressing of whole fruits,

most phytochemicals including polyphenols from arils, seeds and the peels are extracted

23

along with the juice. During industrial juice extraction by hydrostatic pressing the water

soluble peel punicalagins are dissolved in the juice, which contributing to the antioxidant

activity of pomegranate juices (Gil et al, 2000). The predominant polyphenols in

commercial pomegranate juice (PJ) are flavonoids, condensed tannins and hydrolysable

tannins (HTs) (Seeram et al, 2005; Seeram et al, 2006; Gil et al, 2000). According to Gil et

al. (2000), HTs are responsible for approximately 92% of its antioxidant activity. HTs are

classified into gallotannins (1,2,4,6-tetra-O-galloyl-D-glucose and 1,2,3,4,6-penta-O-

galloyl-D-glucose) and ellagitannins (ellagic acid esters of D-glucose with one or more

galloyl substitutions), and are susceptible to enzymatic and non-enzymatic hydrolysis and

are further classified according to the products of hydrolysis. The predominant and the

unique gallagyl ester of pomegranate HTs is punicalagin, which is responsible for about

half the antioxidant activity of the juice (Seeram et al, 2006).

2.3.1 Ellagitannin

The ellagitannins (ETs) are water soluble, hydrolysable polyphenolic compounds (Wilson

and Hagerman, 1990). ETs are nitrogen deficient compounds having molecular weight

ranges from 300 to 20,000 da (Khanbabaee and Van Ree, 2001). ETs are amorphous having

astringent flavor, acts as a weak acids and considered as a secondary metabolites occurs in

cell’s cytoplasm and vacuoles (Khadem and Marles, 2010). Kaponen et al, (2007) had

stated that ETs are formed from gallotannins (GTs) from the oxidative coupling of two

units of galloyl in the carbon 6 and forming one unit of hexahydroxydifenic acid (HHDP)

and generating a monomeric ellagitannin. ETs have ability to form strong complexes with

proteins and polysaccharides due to which it showing important functions in plant

physiology by providing protection against microbial decay (Haslam, 1996). ETs are

24

showing antioxidative property in human diet and prevent cancer and cardiovascular

diseases (Manach and Scalbert, 2004; Madrigal-Carballo et al, 2009). ETs exhibiting

antioxidant activity by the action of absorption and neutralization of free radicals (Due to

redox properties) (Fukuda et al, 2003; Olsson et al, 2004). When ETs are exposed to acids

or bases, then bounds ester gets hydrolyzed and released HHDP group (Fig 2.4). However,

it is a non-stable group requiring a lactonization step to move on to a stable form, leading to

the formation of monomer with high antioxidant activity (Aguilera-Carbo et al, 2008a).

Figure 2.4 Hydrolysis of Ellagitannins (Aguilera-Carbo et al, 2008)

25

2.3.2 Components of Hydrolysable Ellagitannin

Hydrolyzed products of ellagitannin are shown in Table 2.9. The vivid functional

properties shown by pomegranate fruit can be classified based on the parts of the fruit

as mentioned in Table 2.10.

Table 2.9 Components of hydrolysable ellagitannin and their structure (Martin et al., 2009)

Component

name

Molecular structure Molecular

Weight

Description

Punicalagin

1084

gallagic acid + ellagic acid +glucose

Gallagic acid

601 Ellagic acid + ellagic

acid

Ellagic acid

301 gallic acid + gallic

acid

Gallic acid

169 benzene ring with 1

carboxyl group in

position 1 and 3

hydroxyl groups in

positions 3, 4 and 5

26

Table 2.10 Current information on functional properties of different parts of pomegranate

fruit

Part of Pomegranate

fruit

Functional property

Juice Antioxidant (Gil et al, 2000), Anti-atherosclerotic

(Kaplan et al, 2001), Platelet aggregation inhibitor

(Aviram et al, 2000), Anti-cholesterol (Esmaillzadeh

et al, 2004), Anticancer (Nam et al, 2002)

Peel Antioxidant (Singh et al, 2002), Antibacterial,

Wound healing (Murthy et al, 2004), Antitumor

(Castonguay et al, 1997), Antimutagenic (Negi and

Jayaprakash, 2003)

Seed Antioxidant (Singh et al, 2002), Antitumor (Hora et

al, 2003), Estrogenic (Mori-Okamoto et al, 2004)

Anticancer (Kohno et al, 2004).

Pith and carpellary

membrane

Antioxidative, Antibacterial (Kulkarni et al, 2004)

2.3.3 Biosynthesis of Ellagitannins

Information about ETs biosynthesis is limited, however, it is known that the biosynthesis

begins when a glucose molecule forms a complex with a gallic acid (GA) molecule, the

complex is formed in glucose at C-1, forming 1-O-alloylglucose (Salminen et al, 2004).

Later four galloylation consecutive reactions are carried out to form its 1,2,3,4-penta-O-

galloyl-ß-D-glucopiranose (pentagalloyl glucose (PGG). For GTs biosynthesis, this ester

27

group with additional galloyl units forms complex metabolites which can have ten or more

galloyl units (Barbehenn et al., 2004). On the other hand, ETs come from the result of the

molecular oxidations between residues of galloyl and PGG generating the formation of

3,4,5,3',4',5'-hexahidroxidifenol (HHDP), typical unit of ETs; the PGG oxidation is carried

out by action of polyphenoloxidase enzyme (Niemetz and Gross, 2003).

2.3.4 Sources of ellagitannins

As already mentioned, the ETs are precursors of a molecule with high antioxidant capacity.

Presence of ETs has been described in different plant tissues, principally in leaves, stalks

and husks of fruit. The ETs concentration in vegetables is established by the quantification

of ellagic acid (EA) (Swain, 1979). Different part of plant tissues can reveal the different

types of tannins. Young oak leaves contain mainly ETs, while in fresh bark there is a mix

of ellagitannins and pro-anthocyanidins (Lei et al, 2001).

2.3.5 Punicalagin

Punicalagin is a hydrolysable tannins (polyphenolic bioactive molecule) isolated from

pomegranate fruit waste peel, pith and carpellary membrane. This molecule has been

reported in α and β form isolated from pomegranate juice, seed and peels (Gil et al, 2000;

Kulkarni et al, 2004; Seeram et al, 2004). Punicalagins are isomers of 2,3-(S)

hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose. The chemical structure of punicalagin

is shown in Table 2.9. Punicalagin is responsible for pomegranate antioxidant and health

benefits. It can be hydrolyze into smaller polyphenols (lower molecular weight) such as

ellagic acid, gallic acid in vivo. A study has showed that α form of punicalagin act as strong

cancer suppressors (Larrosa et al, 2006; Seeram et al, 2004). Punicalagin has shown

28

remarkable pharmacological activities including antioxidant, anti-inflammatory, hepato

protective (Lin et al, 2001), and anti-genotoxic activity (Chen et al, 2000). A study on

bioavailability of punicalagin in the rats reported that punicalagin and its metabolites were

observed in faeces and urine and also in plasma (Cerda et al, 2003), but the mechanism of

intestinal uptake of punicalagin is unknown.

2.3.6 Ellagic acid

Ellagic acid (EA) have a molecular weight of 301. It is highly thermostable (melting point

359°C) (Bala et al, 2006) due to the four rings that give it a lipophilic property and two

lactones that can act as hydrogen forming and electron acceptors, respectively, which gives

a hydrophilic characteristic (Salminen et al, 2004). Ellagic acid has showed biological

activities as antitumor (Okuda et al, 1993; Castonguay et al, 1997), antiviral (Ruibal, 2003),

antioxidant (Vattem and Shetty, 2002), antimicrobial (Atta-Ur-Rahman et al, 2001) and

anticancer (Losso et al, 2004; Huang et al, 2005). EA can be quantified by

spectrophotometric methods (Khanbabaee and Van Ree, 2001). EA has been proved to be

useful in the cosmetic, pharmaceutical, nutraceutical and beverages industry.

2.3.7. Gallic Acid

Gallic acid is an organic acid (MW 169), also known as 3,4,5-trihydroxybenzoic acid,

found in pomegranate, gallnuts, sumac, witch hazel, tea leaves, oak bark, and other plants.

Gallic acid is found both free and as part of tannins. It is commonly used in the

pharmaceutical industry. Gallic acid can also be used to synthesize the hallucinogenic

alkaloid, mescaline, also known as 3,4,5-trimethoxyphenethylamine. Salts and esters of

gallic acid are termed as gallates (Seeram et al, 2004).

29

2.3.8 Biodegradation of ellagitannin

There are few studies on the enzymatic (fugal enzymes) biodegradation of ellagitannin.

Yoshida et al, (1999) extracted two compounds, novotanin O and P, from Tiouchina

multiflora by enzymatic hydrolysis using tannase enzyme produced by Aspergillus niger.

EA was not released because tannase enzyme did not hydrolyzate HHDP while gallic acid

was released by tannase enzyme activity. Vattem and Shetty (2002) studied enzymatic

hydrolysis of cranberry pomace by using a GRAS fungal strain Lentinus edodes in a solid

state fermentation (SSF). They found an increase in polyphenols concentration and activity

of glucosidase during the fungal fermentation. Huang et al, (2005) studied the enzymatic

hydrolysis of ETs from oak by fungal enzyme (valonia tannin hydrolase) produced by A.

niger SHL 6, results were evaluated only for tannase activity, so it may not sure that

ellagitannin hydrolysis was dr iven by the enzyme. Shi et al, (2005) have studied on

enzyme hydrolysis, they fermented valonia extracts with A. niger and C. utilis and reported

that ellagic acid accumulated due to tannase activity by A. niger and the polyphenol

oxidase by C. utilis. There are need of more research on ETs biodegradation in order to

clearly elucidate its metabolic pathway (Saavedra et al., 2005) with the information

about recovery process of EA. Ascacio-Valdes et al, (2011) reported that tannase enzyme is

responsible for gallotannins hydrolysis and producing GA. Aguilera-Carbo et al, (2007)

reported the degradation of polyphenols (ETs hydrolysis) by a new enzyme different to

tannase from A. niger GH1 resulted into ellagic acid accumulation. Hernandez-Rivera

(2008) has examined a purified enzymatic extract produced in solid state fermentation (by

A. niger GH1) by using polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme can

be responsible for catalyzing of HHDP hydrolysis reaction in ETs to produce ellagic acid,

30

because of that the putative enzyme was named ellagitanin acyl hydrolase or ellagitannase.

However there are requiring more research to clearly understand the mechanisms of

biodegradation of ETs (Scalbert, 1992; Vivas et al, 2004).

2.4 Ellagitannin extraction techniques

There are various extraction temperatures and methodology have been reported for

ellagitannin extraction at temperature ranging from 35 to 100°C from by-products of plant

origin. There are many polyphenols extraction method which are based on different

techniques e.g. solvent (supercritical fluid extraction and pressurized liquid) extraction,

ultrasonic and microwave assisted, solid liquid, maceration extraction etc. has been applied

for extraction of different phytochemicals.

2.4.1 Selection of plant materials

Selection of plant part or material is the most important point before extraction, isolation

and characterization of phytochemicals. Selection of plant part or materal should be

considered on the basis of unexplored plant or plant parts, easily availability in bulk

quantity, teleological importance etc. Selected plant part may be collected and prepared

(hot air drying, grinding etc.) for extraction as required by desirable extraction method

(Kulkarni et al, 2004).

2.4.2 Pressurized Solvent extraction

Pressurized solvents extraction works under high pressure and sometimes temperature also,

which can significantly improve the efficiency of extraction. In solid-liquid extractions,

affinity to solubilize the target phyto-compound should show higher in employed solvent

31

while other compounds should not be solubilizing during extraction (Pronyk and Mazza,

2009). Many researcher has carried out research on optimization of extraction conditions,

e.g. liquid solid ratio, extraction temperature, extraction time, particle size, pressure and

flow rate, to obtain higher recovery of phyto compounds from plant parts (Kaur et al, 2008;

Wijngaard et al, 2012). Conventional method of solid liquid extraction such as Soxhlet

extraction and maceration are time consuming and use large amounts of solvents (Wang &

Weller, 2006). Pronyk & Mazza (2009) has explained that there are two pressurized solvent

extraction methods, which are very popular for extraction (a) Supercritical Fluid Extraction

(SFE) in which CO2 is used as solvent and (b) Pressurised Liquid Extraction (PLE) where

100% water is used, this technique is called subcritical water extraction (SWE).

2.4.2.1 Supercritical Fluid Extraction

SFE technique has been extensively used at laboratory and commercial level. In laboratory

level studies the aim is to recover 100% of the target compound. This technique can be

used to scale up the extraction process to commercial level. There are many examples

where SFE has been up-scaled e.g. decaffeination of coffee beans (Zosel, 1981), extraction

of α-acids from hops to produce hop resins (Bath et al, 1980). Carbon dioxide is mainly

used in this technique for polar target compounds (Diaz-Reinoso et al, 2006). However,

super critical water systems also used to extract polar compounds (Henry & Yonker, 2006).

Critical point of water is 374°C and 22.06 MPa, therefore it may not be used to extract

thermally labile compounds (Lang & Wai, 2001). CO2 has low critical temperature 31.1°C

and a low critical pressure 7.4 MPa. CO2 is safe, food grade and widely available in low

cost and high purity (Reverchon & De Marco, 2006). CO2 as a solvent is slightly polar thus

for polar phytocompounds a co-solvent e.g. ethanol is often added to CO2.

32

2.4.2.2 Pressurized liquid extraction

During pressurized liquid extraction (PLE), pressure is applied with use of temperature

above the boiling point of solvents (Mendiola et al, 2007). Extraction at high temperature

may be advantageous due to increase in the mass transfer rate, extraction rates, increase in

capacity of solvents to solubilize solutes, increase in diffusion rates, and better disruption of

solute matrix bonds, decrease in viscosity of the solvent and decrease in surface tension

(Ramos et al, 2002; Richter et al, 1996). The applied pressure usually ranges from 4 to 20

MPa, which ensures to maintain the solvent in the liquid state at applied temperature.

Extraction of phyto compounds by this technique may requires a proper system to operate

at standard condition.

2.4.3 Non thermal extraction

In addition to PLE techniques discussed above, other techniques exists which may assist

in the extraction of bioactive compounds, such as pulsed electric fields (PEF), ultrasound

waves and microwaves. These techniques could be applied as a preliminary step in the

extraction of bioactive compounds in which due to electrical field on the membrane, pore

formation occurs which enhances cell permeability (Angersbach et al, 2000).

2.4.3.1 Pulsed electric field (PEF) assisted extraction

Traditionally, PEF has been used as a non-thermal preservation technique but extraction of

bioactive compounds from by-products using PEF is not well studied till now. The

technique has been mainly used to extract polyphenols from grape by products. In red

grape by-product anthocyanin level (60%) was increased when PEF was applied as a pre-

33

treatment (1 min at 25°C) (Corrales et al, 2008). In white grape skins pretreatment with

PEF at a temperature of 20°C, recovery of polyphenols was more (10%) than untreated

samples (Boussetta et al, 2009). PEF seems a potential technique but industrial scale

equipment is still under development.

2.4.3.2 Ultrasound assisted extraction (UAE)

UAE can also be used in the food industry to perform extractions. The technique uses high

frequency sound waves (higher than 20 kHz). Various studies has shown to enhance the

extraction of bioactive compounds such as polyphenols from different plant byproducts

Orange peels were treated with ultrasound to extract the flavanones, hesperidin and

naringin. Extraction conditions viz. temperature, solvent ratio and sonication power has

been optimized using RSM (Khan et al, 2010).

2.4.3.3 Microwave assisted extraction (MAE)

Microwaves are electromagnetic waves, which are usually operated at a frequency of 2.45

GHz. Microwaves can access biological matrices and interact with polar molecules such as

water and generate heat. Such rise in temperature generally leads to enhanced extraction

efficiency (Wang and Weller, 2006). The effect of microwave energy and treatment time

increased the content of free phenolics in the extracts and decreased the bound phenolic

content. This indicated that microwave treatments can cleave and liberate the bound

phenolics from the plant tissue. It has been marked by researchers that higher microwave

energy and longer treatment time can results in the degradation of flavonol compounds. The

best advantage of MAE over conventional maceration and solid-liquid extraction is

reduction of extraction time and higher extraction efficiency (Hayat et al, (2010). MAE

34

system may be the costly process for commercial production of polyphenolic compound

from fruit and vegetable byproduct.

2.4.4 Solvent Extraction of polyphenols

Casas et al, (2010) reported that resveratrol, a well-known phenolic compound in grape

byproducts (seed, stem, skin and pomace) was extracted using super critical CO2 at high

pressure (400 bar) and low temperature (35°C) using 5% v/v ethanol as a co-solvent. The

type of pomace from which polyphenols are recovered can influence extraction yields.

Apple and peach pomace exhibits low yields with about 25% of phenols in comparison to

100% using an ethanolic solid liquid extraction (SLE) (Adil et al, 2007). A variety of

polyphenolic compounds have been targeted in super critical CO2 extraction including

resveratrol, naringin and kaempferol glycosides. However, in many cases the efficiency

of extraction is measured not by the levels of specific compounds but as total phenolic

content. It is possible to extract polyphenols from by-products by using other techniques

such as PLE by using suitable solvent.

2.4.5 Types of extraction Solvents

Extraction of phyto compounds from the plant is a trial and error exercise in which

different solvents are tried under different conditions of time and temperature. The success

or failure of the extraction process is monitored by the most appropriate bioactivity assay.

There are various extraction methods for screening the plant for bioactivity like cold

extraction and soxhlet extraction. Selection of extraction methods depends upon the thermal

stability of the target compound. Several organic solvents are listed in Table 2.11 which has

been applied in phyto compounds extraction.

35

Table 2.11 Some commonly used solvents for extraction (George et al, 2001)

Solvent Boiling point oC at 760 mm

n-Hexane 68.7 Carbon tetrachloride 76.8 Benzene 80.1 Chloroform 61.3 Diethyl ether 34.6 Acetone 56.5 Ethanol 78.5 Methanol 64.6 Water 100.0

For the isolation of unknown bioactive molecule from a plant part, the samples are

normally extracted with different type of solvents. Trials of solvents generally done by

sequentially from low polarity to high polarity. Based on polarity of the solvents most

preferred solvent order used for extraction of phytochemicals from plant parts with

unknown composition is as follows (George et al, 2001):

Hexane<Chloroform<Ether<Acetone<Methanol<Water

2.5 Extraction process optimization

2.5.1 Response Surface Methodology for Optimization of Processes

Response surface methodology (RSM) is being used to develop, to improve or to optimize

various extraction and bio-processes (Panda et al, 1997). The statistical design of

experiments consists of three stages, viz. screening of the significant input variables,

modeling the response in terms of the selected variables, and finding the optimal settings of

the variables, using a suitable optimization technique (Khuri and Cornell, 1987).

36

Wang et al, (2013) have studied the optimization of extraction yield and total phenolics

from pomegranate leaves by using RSM based on three level, three variable (solvent

concentration, temperature and time) using Box-Behnken design.

Zhu and Liu (2013) studied optimization of extraction of crude polysaccharides from

pomegranate peel by RSM. To find out the optimal settings of the process variables for

ellagitannin extraction, a model is necessary to predict the various combinations of the

process variables to be optimized. An effective plan of experiment is needed to construct

such model, where the target response (i.e., total ellagitannin content) can be estimated at

different levels of the significant process variables. A full factorial design is not always a

suitable plan of experiment for such a purpose, since, the number of required experimental

runs increases exponentially with the increase of controlled variables. For example, a five

level full factorial design for three factors would require 35 or 243 experiments. However,

there are some reports on the optimization of the process variables for improved

ellagitannin production using a full factorial design of experiments (Wang et al, 2013; Zhu

and Liu, 2013). Tavares et al, (2006) and Vanhulle et al, (2007) have considered two level

variations of the process variables, which assume a linear relation between the response

function and the independent variables, that may not be true in a complex system. The

central composite design (CCD) of experiments is usually used for the purpose (Liu et al,

2009). This design is capable of providing enough number of data points to solve a second

degree polynomial, with a considerably less number of experiments. The polynomial model

provides valuable information about the system, irrespective of whether the response

function has an extremum in the observed domain of process variables or any two process

variables have an interaction effect.

37

Once the response function is known, a response surface (and/or contour plot) can be

generated. For two independent variables, the response surface provides the optimal value

directly. A schematic representation of such a response surface and contour plot is given in

Figure 2.5. However, for more than two variables, only contour plot is not adequate for

finding optimal of the independent variables. Since, the level of other significant variables

influences the contour plot of any two variables. Hence, a suitable optimization technique

should be used to evaluate the optimal settings of process variables for maximization of a

response.

Figure 2.5 Schematic Representations of (a) the experimental space in the Central Composite Design of experiments for two variables and (b) Optimal point in a Response Surface for two independent variables.

38

2.6 Studies on isolation and purification of ellagitannins extract

2.6.1 Techniques of isolation and purification of polyphenols

It is normally very difficult to have a definite procedure or protocol for the isolation and

characterization of bioactive molecules. However, isolation, purification and

characterization of bioactive phytochemicals follows some definite steps like selection and

collection of plant material based on the ethno-botanical information, followed by

extraction using variety of solvents and screening for bioactivity. The crude solvent

extracts were subjected for bioassay of interest. The active extract can then be subjected

for isolation and purification of the active principle (bioactive molecule) responsible for

the bioactivity. Isolation and purification can be achieved by column chromatography

techniques. Isolated compounds can be purified by preparative HPLC or collects the

selective pure compounds after passing through resin packed columns. The purified

compound can be identified by different spectroscopic techniques like UV-Visible, IR,

NMR, Mass spectroscopy followed by functional characterization (Kulkarni et al, 2004).

2.6.2 Purification of ellagitannin extract

Polymeric adsorbent resins are used to purify tannins from an aqueous pomegranate

extract. After adsorption, the resins are subjected to cleansing, gravity evacuation, and

vacuum aspiration of liquid. The adsorbed tannins may be eluted from the resins and the

solvent removed in vacuo to yield a powder of highly concentrated total tannins. The

powder may comprise a high level of ellagitannins, including gallagic acid, punicalagins,

punicalin, ellagic acid, ellagic acid glycosides, anthocyanins, anthocyanidins etc.

39

A suspension of material from the plant is prepared by a variety of methods like blending,

aqueous extraction etc. The plant material may be subjected to enzymatic treatment. The

aqueous solution comprising ellagitannin is applied to a polymeric adsorbent column,

which is then washed with an aqueous buffer to remove unbound material. The tannins of

interest bind to the resin, and may be eluted with a polar solvent, e.g. ethanol, methanol,

acetone, etc. The resin has a surface to which the ellagitannin are adsorbed. A preferred

class of adsorptive resins are cross linked resins composed of styrene and divinylbenzene

such as, AMBERLITE series e.g. AMBERLITE XAD-16, which are available

commercially. It is preferred to use commercially available, FDA approved, styrene-

divinyl-benzene (SDVB) cross-linked copolymer resin (AMBERLITE XAD-16). It has a

macro reticular structure, with both a continuous polymer phase and a continuous pore

phase. Other adsorbents, such as those in the AMBERLITE XAD adsorbent series which

contain hydrophobic macro reticular resin beads, with particle sizes in the range of 100-

200 microns, are also be effective in purification. The AMBER LITE XAD-16 is preferred

since it can be reused many times (over 100 times). Various design may be used for the

purification, including batch adsorption, column chromatography etc. The resins are

washed, e.g. with water or an aqueous buffer to remove unbound material from the extract.

Any solvent (lower alkanols containing 1 to 4 carbon atoms and most preferred is ethanol

(ethyl alcohol). Since it is approved for food use) can be used to remove the adsorbed

ellagitannins. Typically the ethanol is azeotroped with water, however absolute ethanol

can be used. Water containing malic acid and sugars in the pomegranate pass through the

column. These may be collected and can be used in foods as flavors. The eluted tannins

are substantially purified relative to the starting material, and may be further purified, e.g.

40

by chromatography etc. or may be directly used in formulations of interest. (Seeram et al.

2004a).

Thin layer, paper, column chromatographic methods have been used for the separation and

purification of many bioactive molecules. However, due to lack of recovery of sample and

difficulties in quantification, thin layer and paper chromatography were used as an

analytical tool in comparing the different fractions. Column chromatography and TLC

are still widely used due to convenience, economy, availability of variety of stationary

phases for separation of phytochemicals with varying nature and availability of solvents.

Plant materials contain highly complex profiles of phytochemicals; isocratic separation

cannot achieve satisfactory separation. Multiple mobile phases with increasing polarity are

therefore useful for good separation (Kulkarni et al, 2004).

2.7 Studies on physicochemical and functional properties of ellagitannin

Physical, chemical and functional (antioxidant and antibacterial) properties of

phytochemicals were reported by various researcher in crude extract of peel or peel powder.

2.7.1 Characterization of phytochemicals

The process of characterization involves accumulating data from a wide range of

spectroscopic techniques viz. UV-visible, Infra-Red (IR), Nuclear Magnetic Resonance

(NMR) and mass spectroscopy. Each of these techniques gives some basic information

regarding the structure of the molecule. By plotting the changes in absorption against

electromagnetic radiation passed, spectrum can be produced. This spectrum is unique for a

bond in a molecule or for a molecule or a class of molecule. Based on these spectra,

structures of organic molecules can be elucidated. Although almost all parts of the

41

electromagnetic spectrum are used for studying matter in organic chemistry i.e. Ultraviolet

(UV), Visible, Infrared (IR), radio frequency and electron beam (Kemp, 1991a), which are

most often used for structural elucidation (Kulkarni et al, 2004).

2.7.2 UV-Visible spectroscopy

Qualitative and quantitative analysis may be performed in the UV-visible regions to

identify certain classes of compound both in the pure and in biological mixtures. However,

it is used preferentially for quantitative analysis by making use of the fact that

aromatic molecules are powerful chromophores in the UV range. Nature of the compound

can be determined making use of UV-visible spectroscopy (Kemp, 1991b).

2.7.3 Infra-Red Spectroscopy

When Infra-red light is passed through a sample of an organic compound, some of the

frequencies are absorbed, while other frequencies are transmitted through the sample

without being absorbed. A plot of absorbance or transmittance against frequency of the

incident ray is called infrared spectrum. Infrared absorptions are associated with

vibrational changes within the molecule. Infra-red spectroscopy is therefore basically

vibrational spectroscopy. Different bonds (C-C, C=C, C-O, C=O, O-H, N-H etc.) have

different vibrational frequencies, and the presence of these bonds in an organic molecule

can be detected by identifying the characteristic frequency as an absorption band in the

infrared spectrum (Kemp, 1991c; Kulkarni et al, 2004).

42

2.7.4 Mass spectrometry

In the simplest mass spectrometer organic molecules are bombarded with electrons or by

some other means such as laser desorption, converted to highly energetic positively charged

ions (molecular ions or parent ions) which can break up into smaller ions (fragment ions or

daughter ions). The molecular ions, the fragment ions and the fragment radical ions are

separated by deflection in a variable magnetic field according to their mass and charge and

generate an ion current at the collector in proportion to their relative abundance. A mass

spectrum is a plot of relative abundance against the ratio of mass/charge. Using mass

spectrometry, relative molecular weight with high accuracy can be determined and exact

molecular formula with the help of places where molecule preferred to fragment (Kemp,

1991d; Kulkarni et al, 2004).

2.7.5 Antioxidative property of ellagitannin

Free radicals are produced in presence of oxygen during respiration although essential

oxygen for life, also produces a reactive molecules such as superoxide anion, superoxide

radical and hydroxyl radical (O2•, ROO•, OH•) which produce adverse health effects due to

its ability to alter the DNA, lipids and protein oxidation. Over the years, free radicals can

produce genetic alteration of cell division process contributing to the risk of cancer

(Amakura et al, 2000). Overall, the ETs and EA preventing the free radicals formation and

its reaction and damage of cell’s organism (Amakura et al, 2000; Priyadarsini et al, 2002).

43

2.7.6 Clinical properties of ellagitannin

The coordinated reaction between polyphenols and carcinogen benzopyrene-7, 8-diol-9,10-

epoxide (BPDE), this test as chemo preventive agents against cancers caused by

polycyclic aromatic hydrocarbons (PAHs) (Huetz et al, 2005), inhibiting a mutation of

healthy cells, initial step in cancer development (Smith et al, 2004). ETs reduced the

incidence of colon tumors and bladder carcinoma in mice when orally administered (Kellof

et al, 1994). ETs are known for their action to modulate the activation of carcinogenesis by

inhibition of the cytochrome P450 phase I enzyme and inducing the glutathione-s-

transferase involved in the carcinogenic detoxification from phase II. At concentrations

around 1 to 100 mM/L, shows strong anti-proliferative activity against cancer cells in

colon, lung and prostate (Castonguay et al, 1998; Losso et al, 2004). ETs strengthen the

connective tissue and avoid the spread of cancer cells. ETs reduce the negative effects from

chemotherapy and radiation (Varadkar and Dubey, 2001). Reports related to ETs to the

inhibit malignant cells in skin cancer, breast, stomach, lung, esophagus, liver and other

cancer are found in literature (Kaur et al, 2006; Seeram et al, 2006).

2.7.7 Antimicrobial properties of ellagitannin

ETs are reported as antimicrobials because their inhibitory effect against bacteria, fungi and

parasites. It has been observed that some plant extracts from Pteleopsis hylodendron

containing mainly EA derivatives, are active against certain pathogenic bacteria such as

Klebsiella pneumonia, Bacillus cereus, Escherichia coli, Salmonella typhi and Salmonella

pyogenes (Rahman et al, 2001). Extracts from Punica granatum L. showed the ability to

inhibit the growth of Pseudomonas aeruginosa and Bacillus subtilis at concentrations of 70

44

mg/mL (Nascimento et al, 2000). Fractions obtained from extracts of Punicalagin showed

a clear inhibition zone of 20 mm of susceptibility against strains of methicillin-resistant

Staphylococcus aureus (MRSA), at a minimum inhibitory concentration (MIC) of 61.5

µg.ml-1 (Machado et al, 2002). By purification of pomegranate peels extracts (Seeram et al,

2005), punicalagin, punicalin, gallagic and ellagic acid which exerted an effect on the

growth of bacteria such as E. coli, Candida albicans and Crypotococcus neoformans as

well as the fungus Aspergillus fumigatus, were obtained (Nascimento et al., 2000). The

phenolic compounds extracted from pomegranate have the ability to inhibit both gram

negative and gram-positive bacteria (Naz et al, 2007), as well as have bio-insecticide

activity (Abo-Moch et al, 2010). The ellagitannin tellimagrandin II; extracted from a

Brazilian popular plant (Ocotea odorifera) used for the cure of skin exhibited potent

inhibitory activity against the yeast C. parasilopsis with a MIC of 1.6 ml. The use of EA at

different concentrations in edible films for subsequent application in avocados reduces

changes in the appearance of solid content, pH, aw, lightness and weight loss;

furthermore, maintains quality and extends shelf life of avocados inoculated with

high loads of the fungus Colletotrichum gloeosporioides (Saucedo et al, 2007). In

addition, the use of extract with 13% of EA inhibits in vitro growth of Propionibacterium

acnes and Staphylococcus epidermis, which cause skin diseases like acne

(Panichayupakaranant et al, 2010).

2.7.8 Toxicological studies of ellagitannin

Aqueous pomegranate fruit extracts has not shown toxic effect in mice treated with

(Amorim, et al, 1995). The pomegranate extract showed low cytotoxicity in vitro and in

vivo, without mutagenic effects in mice (Yamaguchi et al, 2011). It has been found that a

45

diet containing 6% punicalagin given to rats for 37 days caused no obvious toxicity (Cerda

et al, 2003). The oral LD50 of a pomegranate extract standardized to 30% punicalagins, 5%

ellagic acid, and 0.3% gallic acid (photometric assessment 70% polyphenols, trade name

POMELLA) was found to be greater than 5 g/kg body weight in rats and mice. The “no

observed adverse effect level” (NOAEL) was defined as 600 mg/kg body weight/day of

pomegranate extract (Patel et al, 2008).

Pomegranate fruit extract exerted an embryo protective effect against adriamycin induced

oxidative stress in 12 days old chick embryos. After 24 and 48 h of incubation, 70 µg/egg

of adriamycin on its own produced a significant dose versus time-dependent reduction in

body weight and volume of amniotic fluid and a dose related increase in gross

embryological deformities and significant changes in the levels of biochemical markers in

amniotic fluid. These changes were significantly reduced by pre administration of

pomegranate fruit extract at a dose of 200 µg/egg (Kishore et al, 2009). In other tests,

pomegranate extract was found to be protective against methotrexate induced oxidative

bone marrow damage (Sen et al, 2014).

2.8. Stability of ellagitannin

Bakowska et al, (2003) stated that polyphenolic compounds are very unstable and highly

susceptible to degradation. The stability of polyphenols under different conditions is a very

important aspect which has to be taken into account to ensure that phenolic compounds

have the desired properties and maintain their activity and structure during the different

stages of processing, which can involve high temperatures, light, oxygen, solvents, the

presence of enzymes, proteins, metallic ions, or association with other food constituents

46

(Ovando et al, 2009). The thermal stability of polyphenols is crucial and may be correlated

with both the extraction and characterization methods, and the recommendations on their

fields of use. Volf et al, (2013) reported the stability of gallic acid, catechin, and vanillic

acid at different temperatures. The data obtained showed a good correlation with the UV

stability. Catechin was the most unstable compound, gallic acid and vanillic acid exhibited

their degradation rates almost similar at 60oC and 80oC, with degradations of 15% and

25%, respectively. On the other hand, at 100oC over 4 h of exposure, the degradation of

gallic acid (30%), catechin (32%), and vanillic acid (37%) was observed.

2.9 Application of ellagitannin

A probiotic product was developed by incorporating pomegranate juice in the formulation

of probiotic yoghurt (Arjmand, 2011). Polyphenols (70%), trade name POMELLA,

BANNERBIO, pomegranate husk extract is available in international market. Due to

antioxidative, colouring, antibacterial properties of ellagitannin, it can be used in foods,

drugs and cosmetic products as an active components (Seeram et al, 2004). Gonzalez-

Molina et al, (2009) has developed the new drink made up of 75% of pomegranate juice

and 25% of lemon juice (v:v), has emphasized by its high antioxidant capacity due to

presence of punicalagin isomers, anthocyanins and vitamin C.

2.9.1 Sharbet

Sharbet is a sugar syrup based drink in India. Term Sharbat is originated from Persian and

Sherbet from Turkish. It is known as cognate to syrup in British and American English.

Historically it was a cool, iced fruit soft drink. It is sweet and served chilled. Popular

47

sharbet are made from rose water, sandal wood, bael, lemon, mango, pineapple falsa

(Grewia asiatica) etc. Most of the sharbet are very common in Indian, Turkish,

Iranian, Arab, Afghan, Pakistani, and Bangladeshi homes. The meaning, spelling, and

pronunciation have fractured between different countries. It is usually spelled as “sharbet”

or “cognate to syrup”

FSSAI (Food Safety Standard Authority of India), India has explained that the Sharbet or

Synthetic Syrup means the syrup obtained by blending syrup made from sugar, dextrose or

liquid glucose. It may also contain fruit juice and other ingredients appropriate to the

product. It shall be free from burnt or objectionable taints, flavours, artificial sweetening

agents, extraneous matter and crystallization. It may contain citric acid, permitted colours,

permitted preservatives and permitted flavouring agents.

2.9.2 Lemon

Lemon was initially used as an antiseptic and an antidote for various poisons. It is probably

one of the most valuable citrus fruits, inexpensive and easily available, lemons can be

added to a variety of recipes such as lemon cakes, lemon chicken and beverages such as

lemonade.

2.9.3 Health Benefits of Lemon

Lemon is effective against nausea, heartburn and parasites. It works best when the juice is

diluted with hot water. Lemon in water is a wonderful remedy for people with heart

problems. It is known to reduce mental stress and depression. Diluting lemon juice with

lukewarm water and honey is a popular choice to weight loss. Lemon juice massaging on

the gums of teeth stops bleeding and removes bad odour. Respiratory and breathing

48

problems are also cured with lemon juice. Being a rich source of vitamin C, it helps in

dealing with asthma. It is an excellent fruit for fighting throat infections and tonsillitis.

Daily consumption of lemon water acts as an anti-ageing remedy.

Findings from review of literature

• Pomegranate fruit is an important fruit having many medicinal properties, being

cultivated in different part of world.

• Pomegranate fruit production in India is 1.3 million MT during 2013-14

• Among Indian states, Maharashtra is leading state in terms of pomegranate cultivation

and production.

• The Bhagwa variety is very common and abundant variety among all varieties grown in

India. Hence this variety selected for research purpose.

• The average price of pomegranate is Rs. 80-100 per kg. in the market of Indian states.

• In India 90% pomegranate fruit is generally consumed in fresh form.

• Fresh whole pomegranate fruit consisting of 31-39% peel, pith & membrane part, 50-

60% aril part and aril can produce up to 80% juice & pulp and 20 % woody seeds as by

product.

• Pomegranate is having many phytochemicals i.e. polyphenols (flavonoids,

anthocyanins, condensed tannins & hydrolysable tannins), organic and phenolic acids,

sterols and triterpenoids, triglycerides and alkaloids.

• Pomegranate fruit peel, pith and membrane part remained as waste after juice extraction

which is having valuable polyphenolic compounds i.e. punicalagin, ellagic acid and

gallic acid. These commonly known as ellagitannin.

49

• Ellagitannins (ETs) are water soluble polyphenolic compounds MW ranges from 300 to

20000 da, having astringent flavor, acts as weak acids and considered as a secondary

metabolites occurs in cell cytoplasm.

• ETs are formed from gallotannins (GTs) from the activated coupling of two unit galloyl

in the carbon-6 and forming one unit of hexahydroxy difenic acid (HHDP) and

generating a monomeric ellagitannin.

• ETs are showing important function in plant physiology by providing protection against

microbial decay, due to ability of ETs to form strong complexes with proteins and

polysaccharides.

• ETs are showing anti-oxidative property by the action of absorption and neutralization

of free radicals in human diets and doing the prevention of cancer and cardiovascular

diseases.

• Punicalagin (MW 1084), ellagic acid (MW 301) and gallic acid (MW 169) are found in

purified peel extract.

• RSM would be a useful technique for the improvement of ellagitannin production by

physical, chemical and enzymatic method with specific variables and their ranges, when

selected appropriately.

• There is no published report on the optimization of the extraction condition and various

extraction methods for enhanced ellagitannin production from pomegranate peel by

using RSM. Such information is important for large scale ellagitannin production by

using a suitable method and conditions.

• Purification of plant polyphenolic extract can be done by Amberlite XAD-16 packed

column (FDA approved) chromatography, subsequently elution with organic solvents

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e.g. ethanol, methanol acetone etc. and drying of eluate under low pressure and

temperature to get the ETs powder.

• Physico-chemical and thermal properties of ETs powder are not yet explored very well.

In few studies NMR, LC-MS, FTIR characterization has been reported for a particular

part of pomegranate fruit.

• Extraction of polyphenolic compounds from plant’s parts has been reported, on the

basis of that polyphenol extraction method could of three type i.e. physical, chemical

and enzymatic or fermentation. These extraction method has been applied separately

not as comparative analysis to explore the efficiency of methods.

• There is no study founds about statistical design (e.g RSM) of experiments for

extraction of ellagitannin from pomegranate peel, while applying physical, chemical

and enzymatic methods simultaneously in terms of quality and quantity of ETs.

• Antibacterial and toxicological property of ETs has been studied at some specified

condition and method only. These properties may be enhanced during extraction when

applying different condition and method, hence it becomes necessary in view of safety.

• There are few studies on stability of ETs at very general conditions. Hence it is

necessary to examine the storage stability of ETs powder under different storage

conditions and in economical packaging.

• Pomegranate juice has been applied as neutraceutical, antioxidative agent in dairy

product (e.g. yoghurt) and mixed with other fruit juice (lemon juice). Ellagitannin

containing commercial product (POMELLA) is available in International market but

ETs still remains unexplored for many foods (ingredients, semi processed and

processed foods) deficient in polyphenolic content.

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