CHAPTER 1 INTRODUCTION, SIGNIFICANCE, AND OBJECTIVES. Pomegranate (Punica granatum) is an important fruit belongs to the botanical family,

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1 CHAPTER 1 INTRODUCTION, SIGNIFICANCE, AND OBJECTIVES 1.1 INTRODUCTION Pomegranate (Punica granatum) is an important fruit belongs to the botanical family, Punicaceae. It is cultivated mainly in Iran, India, Spain, (South Eastern), Israel, United States. In India, it grows in wild form in submountaineous and outer Himalayas of Himachal Pradesh, Jammu, Kashmir and Uttrakhand. According to National Horticulture Board of India (2014), the pomegranate cultivation area and production is 131thousand ha and 1346 thousand MT respectively. Maharashtra is the leading producer of pomegranate followed by Karnataka, Andhra Pradesh, Gujarat and Tamil Nadu. Bhagwa variety among all the varieties grown in India, is easily available on commercial scale. This is a medium size fruit having soft seeds, reddish flesh and sweet juice. Considering world market for pomegranate, India has a great opportunity to contribute in international market as a counter-season producer and exporter. There are many types of pomegranate like the double flowering, ornamental type, and a white flowered type (poor fruit quality) are cultivated in India. They have the sugar content between 12 and 16 per cent and they contain considerable amount of vitamins, polysaccharides, polyphenols (tannin) and important minerals (Al-Maiman & Ahmad, 2002). Mahajan et al, (1992) reported its richness in vitamin C, minerals e.g. Calcium (Ca), zinc (Zn), manganese (Mn) and its usefulness for making various digestive and other medicines. Legua et al, (2012) stated 1

2 that the genotype factor should be considered as the most influencing factor in future breeding programs to enhance the synthesis of beneficial bioactive compounds e.g. organic acid profiles, total phenolics. Pomegranate fruits are widely consumed in fresh form or as juice, jam, jellies and wine and also as flavoring and coloring agent. The juicy outgrowth of pomegranate seed (aril) is the edible part of the fruit, which gives delicious juice. The sour aril when dried along with the seed gives the anardana, generally used as condiment. The kernels are also used for garnishing dessert and salads (Al-Maiman and Ahmad, 2002). The demand for pomegranate juice (PJ) worldwide has increased significantly in recent years (Zhou et al, 2011). Seed, peel and membrane part of pomegranate are generally considered as waste material after juice extraction. The potent antioxidant capacity of pomegranate peel powder or its extract against lipid peroxidation may be the central link for the antiatherogenic effects of pomegranate peel powder or its extract on lipoproteins (Labib and Hossin, 2009). In the last few years the identification and development of phenolic compounds or extracts from different plants has become a major area of health and medical related research (Dai and Mumper, 2010). Pomegranate peel and peel extract are well known for its ethno medical relevance and chemical features, the biomolecule available have been proposed, as substitutes of synthetic food additives, neutraceutical and chemo preventive agents. (Akhtar et al, 2015). It has been reported that pomegranate juice and pomegranate husk (peel) extracts exhibit potent biological properties, i.e., anti-atherogenic effects in human that may be due to the presence of polyphenols known as ellagitannins. Pomegranate fruit husk (peel) is a rich source of hydrolysable tannin called ellagitannins (ETs), an antioxidant. ETs may serve in the prevention of cattle diseases and improvement of meat products, which making it an 2

3 attractive component in cattle feed (Shabtey et al, 2008). Pomegranate peel is very rich in ellagitannins such as punicalagin, punicalin, gallic acid (GA), ellagic acid (EA) and ellagic acid glycosides (Hexoside, pentoside, rhamnoside etc.) (Cerda et al, 2003, Gil et al, 2000, Lu and Yuan, 2008). Punicalagin has generated particular interest with regard to human health including antioxidant activities (Lin et al, 2001 and Tzulkar et al, 2007). In the commercial pomegranate juice industry, ETs extracted from the husk in significant quantities. Pomegranate husk a byproduct of the pomegranate juice industry is therefore an inexpensive and abundant source of ETs. Pomegranate ellagitannin has been identified as the active antioxidant compounds having anticancer activities, protecting low density lipoprotein, cholesterol from oxidation in- vivo, (Seeram et al, 2004). Pomegranate peel extract are also being investigated for their potential use as food bio-preservatives and for the formulation of product in the neutraceutical industry. The pomegranate extracts also used as a source of ellagic acid derivatives, including hydrolysable ellagitannins, for use in-vitro and in-vivo biological studies. This is an useful peel for industrial applications and could provide a low-cost means to use a currently underutilized fruit by-product to develop phytoceuticals with potential health benefits and to develop products for use in the cosmetic and food bio-preservative industries. Importance of the natural antioxidant for medical and food application has been reported by Spigno et al., (2007). Increasing the use of juice industry waste (such as juice and wine making waste) leads to low cost production of antioxidant (phenolic compounds). Recovery of these components is commonly performed through a solvent extraction, but at present sufficient data on the method and conditions for extraction is not available. 3

4 Kulkarni et al, (2004) isolated and characterized the phenolic compounds, punicalagin ellagic acid and gallic acic from the ethanol extract of pith and capillary membrane of pomegranate fruit. These compounds exhibited potent DPPH (2, 2-Diphenyl-1- Picrylhydrazyl) radical scavenging activity (Liu et al, 2011). Moreover, results are difficult to compare because phenols are measured in different ways and sometimes only the phenolic content of the final extract is reported but not the total yield. The aim of an extraction process should be the maximum yield of compounds with the highest quality (concentration of the target compounds and antioxidant power of the extract). The effect of drying before extraction and processing parameters on the extraction kinetics and product properties were studied using water as a solvent for the extraction. The yield and content of antioxidants increased when increased water sample ratio and temperature but antioxidant activity was low at high extraction temperature (Qu et al, 2010). Robledo et al, (2008) reported that pomegranate husk is a good support and an excellent substrate for the production of high commercial interest metabolites like ellagic acid due to the degradation of ellagitannin. Production of ellagic acid may be high due to biodegradation (microbial enzymes activity) of ellagitannin (Carbo et al, 2007). Total hydrolysable polyphenols of pomegranate peel were degraded during the first 72 hours of solid state fermentation by Aspergillus niger culture (GH1and PSH strains), probably by tannin acyl hydrolase activity and produced pure ellagic acid in the fermented mass which was extracted by simple extraction process. The antimicrobial activity of pomegranate fruit peel extract was evaluated by using in vitro and in situ methods against food borne pathogen viz. Listeria monocytogens, Staphylococcus aureus, Escherichia coli and Yersinia enterocolitica and 4

5 phytochemical analyses revealed the presence of active inhibitors in peels including phenolic and flavonoids (Al-zorkey, 2009). Kulkarni et al, (2004) are also found that punicalagin is the major component responsible for antioxidant and health benefits of pomegranate juices and peel extracts. Punicalagin is the largest molecule found intact in rat plasma after oral ingestion and were found to show no toxic effects in rats who were given a 6 per cent diet of punicalagins for 37 days. Patel et al, (2008) also investigated the toxicological effect of ellagitannin and concluded that no observed adverse effect level (NOAEL) for standardized pomegranate fruit extract when the highest dose was determined as 600 mg/kg body weight/day. Research has shown that the synergistic combination of punicalagin with other minor pomegranate ellagitannins, specifically punicalin and gallagic acid, results in superior biological activities. Extracts comprising these compounds and extracts comprising total pomegranate tannins are useful for a variety of applications including pharmaceutical, neutraceutical and cosmetic uses. Seeram et al, (2004) have emphasized that pomegranate derived foods are widely consumed and there have been conflicting reports regarding the toxicity of punicalagin anomers (punicalagin, punicalain, gallagic acid and ellagic acid). There is a need for in depth in-vitro and in-vivo studies to determine the biological properties of these compounds. Unfortunately there are no cost effective and rapid methods available to obtain pomegranate ETs in sufficient yields and purity required for in- vitro and in- vivo biological studies. Anna and Paul (2011) have studied the stability of encapsulated polyphenols at 8 o C and 25 o C was evaluated for 12 months storage and found better protection for phenolics during 5

6 storage. Radical scavenging activity studies demonstrated significant antioxidant activity of microencapsulated powders before and after storage. Volf et al, (2013) studied the thermal stability, antioxidant activity and ultraviolet light stability of standard polyphenols solutions e.g. catechin, gallic acid, vanillic acid and found that phenolic compounds were relatively stable. Li et al, (2015) quantified eight monophenols (including gallic acid, punicalagin, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and ellagic acid) in the pomegranate peel to interpret the consistency of the quality test. Extract of pomegranate is also Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Mustafa et al, (2014) have been used the ellagitannin compounds in dairy products and reported that more than 75% of the panelists accepted the phenolic enriched ice creams in sensory evaluation, which lends supports to such products for commercial introduction to the general public with the potential as functional foods. 1.2 SIGNIFICANCE OF THIS WORK From the critical analysis of published literature and relevant information, the following points bring out the significance and salient features of the present investigation: Utilization of peels, pith and carpellary membrane which remains as waste after extraction of juice from pomegranate fruit. After separation of edible arils from fruits, the peel part (48-50 %) goes as waste. That means if one kg fruit cost is Rs.100, peel of worth Rs goes waste such loss attracts attention to investigate the utilization of pomegranate peel. 6

7 This study explored the prospects of utilization of pomegranate peel as cattle feed after extraction of ellagitannin from fresh pomegranate peel. In this study three extraction methodologies i.e physical, chemical and enzymatic (enzymes as well as microbial source of enzymes) were applied and carried out a comparative study at different time temperature combinations to find an optimal time and temperature of extraction by using Response Surface Methodology. This study suggested a cost and energy saving method and optimized condition to produce purified ellagitannin powder from fresh pomegranate peel. The present research work includes study of the physicochemical, thermal, antibacterial and toxicological properties of purified ellagitannin powder extracted from pomegranate peel. This study suggested suitable storage condition (time and temperature) and suitable packaging material for handling the purified ellagitannin. Uses of purified ellagitannin powder as an additive (source of polyphenol/antioxidant) in sharbet (a common ready to serve beverage, deficient in polyphenols) or cognate to sugar syrup. 7

8 The objectives of the present research have been outlined on the basis of significance. 1.3 OBJECTIVES The objectives of this research were as follows: 1. Comparative study on the effect of extraction methods (physical, chemical, enzymatic) on quality and yield of total ellagitannin content extracted from pomegranate peel. 2. Isolation, purification of peel extract and preparation of ellagitannin powder. 3. To study physico-chemical, antimicrobial and toxicity properties of the ellagitannin powder. 4. Studies on storage stability of the ellagitannin powder under different conditions of temperature and packaging material. 5. Application of ellagitannin powder in sharbet and its acceptability by sensory panel. 8