Tariq Ismail - Higher Education Commissionprr.hec.gov.pk/jspui/bitstream/123456789/7574/1/Dr... · M.Sc. (Hons.) Food Technology A Thesis Submitted in Partial Fulfillment of the Requirements

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Pomegranate Peel Based Novel Food Product

By

Tariq Ismail 2010-ag-241

M.Sc. (Hons.) Food Technology

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

In

Food Science and Technology

INSTITUE OF FOOD SCIENCE & NUTRITION

FACULTY OF AGRICULTURAL SCIENCES & TECHNOLOGY

BAHAUDDIN ZAKARIYA UNIVERSITY

MULTAN, PAKISTAN

2016

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3

To,

Incharge Examinations,

Bahauddin Zakariya University,

Multan.

“I the supervisor certify that the contents and form of thesis submitted by Mr.

Tariq Ismail Regd. No. 2010-ag-241, have been found satisfactory and recommend

that it may be processed for the award of degree of PhD in Food Science &

Technology”.

1. Supervisor:

(Dr. Saeed Akhtar)

2. Ex Officio Member:

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Declaration

I hereby declare that all the contents of the thesis, “Pomegranate peel based novel

food product” are product of my own research and no part has been copied from any

published source (except the reference standard, mathematical or genetic models, equation,

formulae, protocols etc). I further declare that this work has not been submitted for award of

any other diploma/degree. The university may take action if the information provided is

found inaccurate at any stage. (In case of any default the scholar will be proceeded against

per HEC plagiarism policy).

Tariq Ismail Regd: No. 2010-ag-241

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Dedicated

To

My Beloved Parents

&

My Source of Inspiration

“Dr. Saeed Akhtar”

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Acknowledgement

All praises and thanks are for the almighty Allah, the ultimate source of knowledge and

wisdom, the creator, the dominant, the most supreme, the self-existing and all sustaining, whose

grace and mercy enabled me to accomplish this piece of work. Praise for the Holy Prophet

(SAW) for enlightening our souls with faith in almighty Allah.

Indeed the research conducted and work described in this thesis was made possible by the

contributions of knowledgeable experts, sincere colleagues and fellows and the institution to

which I am really indebted. I offer my deepest gratitude to all respectable that all the way

encouraged and supported me in terms of their valuable time and intellect to accomplish the task.

I have no words that fully convey the sense of immense and deep gratitude to my supervisor Dr.

Saeed Akhtar, Director, Institute of Food Science and Nutrition, Bahauddin Zakariya University

Multan for accepting me as his first student, unprecedented support and guidance in project

designing, technical assistance in research and write up and, to well in time management of

project resources. My heartfelt thanks to Prof. Dr. Piero Sestili, I feel really proud to have time

to time guidance in various aspects from such a knowledgeable and courteous personality.

I feel great pleasure to express my sincerest feelings of gratitude to my teachers and colleagues

Dr. Muhammad Riaz, Dr. Muhammad Tauseef Sultan, Dr. Ahsan Sattar Sheikh, Dr.

Aneela Hameed, Mr. Amir Ismail, Dr. Majid Hussain, Mrs. Mamoona Amir, Mr. Khurram

Afzal, and Mr. Tanweer Aslam for their technical assistance, valuable comments and sound

suggestions to improve the quality of work.

I feel delighted to acknowledge the contribution of my dear students and friends particularly

Khalid Amin, M. Saad Hashmi, M. Umair Rafiq, M. Arif Shahzad, Khurram Muaz, Zeeshan Ali

Shah, Muhammad Qamar Saleem, Raheel Suleman, Talha Ali Khan, Iram Ashraf, Sidra Naureen

and Komal Akhtar. Sincere thanks to laboratory and office staff to facilitate in laboratory and

office associated matters.

Warm thanks to my brothers, Amir and Kashif; my sisters Attia amir, Hafsa and Javeria; my

angels Eshal and Iffra, and my better half for their limitless affection, prayers and understanding

when I could not be able to give good time and attention to my family.

Last but not least, my deepest gratitude for Maan g & Abu g for endless support, moral strength,

material welfare throughout my life. Indeed, your love, encouragement, support and prayers

made a big difference in my life.

Tariq Ismail

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ABSTRACT

Pomegranate peel (PoP) is a valuable waste of juice processing industry and is a rich source

of biomolecules of immense health significance. Plentiful research based evidences on

ethnopharmacological and nutraceutical properties of PoP calls for the attention of food

scientists in designing food products with innate ability to improve quality of life. This study

focuses on exploring nutritional and nutraceutical properties of PoP, hydro-alcoholic extracts

and the bagasse as ingredient of choice in developing novel food product. An indigenous

cultivar of pomegranate “Alipuri” was procured from the local farm of Southern Punjab and

pomegranate peel hydro-alcoholic extracts (PoPx) were derived from the peel fraction of the

fruit. Nutritional and biological properties of PoP revealed the waste as a potential source of

compounds bearing significant antimicrobial, antioxidant and antiulcer activities. Hydro-

alcoholic extracts of PoP revealed significantly (p<0.05) higher concentration of phenolics

i.e. 427.19 mgGAE/g and radicals scavenging activities as measured by DPPH and FRAP

assays. Hydro-alcoholic extracts of PoP inhibited urease activity by 97.9% and IC50 for

different extracts was recorded ~44.4 µM. PoP extracts presented significant inhibitory

properties against gram positive and negative microorganisms including Pseudomonas

aeruginosa, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Staphylococcus

aureus and Aspergillus niger. Minimum inhibitory concentration (MIC) of PoPx was

recorded between 0.25–0.89 mg/mL. With reference to its features to be utilized as a food

ingredient, PoP and the peel bagasse (PM) were incorporated in baked good for the

development of nutritionally enriched and biologically stable cookies. The study revealed

PoP supplementation to impart significant (p<0.05) improvement in dietary fibers (0.32–1.96

g/100 g), total phenols (90.7–161.9mg GAE/100 g) and inorganic residues (0.53–0.76 g/100

g) of cookies. Cookies carrying highest levels of PoP (7.5%), PoPx (1.0%) and PM (7.5%)

were recorded with more than 50% radical scavenging activity whereas, products phenolics

pool significantly inhibited oxidative and microbiological deterioration of the finished good

during three months storage period. Cookies supplemented with PoP were recorded to

present organoleptically acceptable quality at 6.0% supplementation while least difference in

sensorial profile was noticed among control and cookies developed with 1.0% PoPx. Toxicity

assessment of PoPx at higher doses i.e. 1000ppm presented non-lethal response in brine

shrimps larvae. Safety assessment of PoP, PoPx and PM revealed non-significant (p>0.05)

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effect of supplementation on various blood indices among normal healthy rats model. Lipid

modulatory role of PoP and its extracts was witnessed convincingly indicating PoP and the

extracts as viable ingredient in improving high density lipoprotein and lowering low density

lipoprotein concentration. The study delineates no abnormal rise or fall in hematological

parameters thereby suggesting a nontoxic response of PoP and its various fractions. The

findings of this research propose PoP as a valuable waste of food processing industry that

could further be exploited in designing nourishing recipes bearing disease preventive and

ameliorative features.

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Contents

Chapter 1 Introduction ................................................................................................ 13

Chapter 2 Review of Literature ..................................................................................... 19

2.0 Preamble ............................................................................................................. 19

2.1. Pomegranate peel phytochemistry ..................................................................... 19

2.2 Structure and activity relationship of PoP phenolics ......................................... 21

2.3 Traditional medicinal uses ................................................................................. 22

2.4 Peel phenolics extraction modeling.................................................................... 22

2.5 Bioactivities of pomegranate peel phenolics ...................................................... 24

2.6 Nutraceutical properties of pomegranate and peel extracts ............................... 26

2.6.1 Cardiovascular protective role ..................................................................................26

2.6.2 Anti-inflammatory and anti-allergic properties ........................................................27

2.6.3 Anticancer perspectives ............................................................................................27

2.6.4 Antimicrobial potential of peel extracts .............................................................................31

2.6.5 Anti-influenza and anti-malarial responses ..............................................................32

2.6.6 Wound healing potential ...........................................................................................33

2.6.7 Pomegranate peel extracts and oral pathogens ....................................................................33

2.7 PoP and PoPx - a biological class of food additives .......................................... 34

2.7.1 Antioxidants potential of PoP and PoPx .................................................................35

2.7.2 PoP and PoPxas dietary supplements .......................................................................36

2.7.3 Stabilization of unsaturated fatty acids in food systems ...........................................37

2.7.4 PoP and PoPx as barriers to food spoilage and infections ........................................38

2.7.5 PoP enhances functional quality of foods .................................................................39

2.7.6 PoP and PoPx as prebiotics .......................................................................................40

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2.8 Functional and Toxicological Levels of PoP and PoPxFor Food Uses.............. 41

2.9 Limitations and future directions ....................................................................... 42

Chapter 3 Materials and Methods .............................................................................. 44

3.1 Procurement of Raw Material ............................................................................ 44

3.2 Preparation of Plant Material ............................................................................. 44

3.3 Chemical Analysis of Peel ................................................................................. 44

3.3.1 Proximate Composition ............................................................................................44

3.3.2 Estimation of Minerals ........................................................................................................47

3.3.3 Peel antioxidants profiling ....................................................................................................47

3.3.4 Antioxidant Assay .....................................................................................................48

3.3.5 Antimicrobial assay of pomegranate peel extracts ...................................................49

3.3.6 Urease Inhibition Activity.........................................................................................50

3.4 Product Development ......................................................................................... 50

3.5 Cookies ............................................................................................................... 51

3.5.1 Preparation of cookies...............................................................................................51

3.5.2 Sensory evaluation ....................................................................................................52

3.6 Product stability study ........................................................................................ 52

3.6.1 Microbiological quality of cookies ...........................................................................52

3.6.2 Free fatty acid levels of cookies................................................................................53

3.6.3 Total phenolics and antioxidant activity of cookies extracts ....................................53

3.6.4 Lipid Peroxidation by Thiobarbituric acid Assay .....................................................53

3.7 Toxicological evaluation of pomegranate peel, meal and peel extracts ............. 54

3.7.1 Cytotoxicity/ brine shrimps lethality test ..................................................................54

3.8 Efficacy study ..................................................................................................... 54

3.8.1 Treatments.................................................................................................................55

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3.8.2 Procurement of rats ...................................................................................................55

3.8.3 Experimental design..................................................................................................55

3.8.4 Preparation of diet .....................................................................................................55

3.8.5 Physical parameters ..................................................................................................57

3.8.6 Sampling procedure ..................................................................................................57

3.8.7 Plasma analysis .........................................................................................................57

3.8.8 Hematological Analysis ............................................................................................63

3.9 Statistical Analysis ............................................................................................. 63

Chapter 4 Results and Discussions......................................................................... 64

4.0 Preamble ............................................................................................................. 64

4.1 Extracts yields and total phenolic contents of pomegranate peel fraction ......... 64

4.2 Free radicals scavenging properties of PoPx...................................................... 68

4.3 Urease inhibitory/antiulcer properties of PoPx .................................................. 69

4.4 Antimicrobial properties of PoPx ....................................................................... 70

4.5 Cytotoxic effects of PoPx in brine shrimps ........................................................ 74

4.6 Nutritional composition of pomegranate peel and peel supplemented cookies . 76

4.6.1 Nutritional profile of PoP and PoP supplemented cookies .......................................76

4.6.2 Mineral profiling of PoP and PoP supplemented cookies ........................................79

4.6.3 Total phenolics and antioxidant capacity of PoP supplemented cookies .................81

4.7 Cookies stability study - free fatty acid levels ................................................... 84

4.8 Organoleptic evaluation of PoP supplemented cookies ..................................... 86

4.9 Effect of supplementing PoPx and PM in Cookies ............................................ 89

4.9.1 Nutritional composition of PoPx and PM supplemented cookies ............................89

4.9.2 Total phenolics and antioxidant potential of cookies ...............................................91

4.10 Microbiological stability of cookies ................................................................... 93

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4.11 Oxidative stability of cookies ............................................................................. 95

4.11.1 Thiobarbituric acid contents of PoPx and PM supplemented cookies ......................95

4.11.2 Effect of PoPx and PM supplementation on free fatty acids contents of cookies ....96

4.12 Organoleptic acceptability of PM and PoPx supplemented cookies .................. 99

4.13 Safety Evaluation of Pomegranate Peel, extracts and Peel Meal ..................... 103

4.14 Effect of PoP, PM and PoPx on feed intake and feeding patterns of rats ........ 104

4.14.1 Effect of PoP, PM and PoPx supplementation on feed intake (g) in male albino

wistar rats ..............................................................................................105

4.14.2 Effect of PoP, PM and PoPx supplementation on weight gain (g) in male albino

wistar rats ..............................................................................................107

4.14.3 Effect of PoP, PM and PoPx supplementation on protein intake (g) in male

albino wistar rats ...................................................................................109

4.14.4 Effect of PoP, PM and PoPx supplementation on protein digestibility (%) in

male albino wistar rats ..........................................................................111

4.14.5 Effect of PoP, PM and PoPx supplementation on Feed Conversion Efficiency

(FCE) in male albino wistar rats ...........................................................113

4.14.6 Effect of pomegranate peel meal supplementation on organ to body weight of

normal albino wistar rats .......................................................................115

4.14.7 Effect of pomegranate peel powder supplementation on organ to

body weight of albino wistar rats ..........................................................117

4.14.8 Effect of pomegranate peel extracts supplementation on organ to body

weight of albino wistar rats ...................................................................119

4.15 Serum Chemistry .............................................................................................. 121

4.15.1 Effect of feeding PoP, PoPx and PM on serum triglycerides levels of albino wistar rats 121

4.15.2 Effect of feeding PoP,PoPx and PM on serum total cholesterol levels of albino

wistar rats ....................................................................................................123

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4.15.3 Effect of feeding PoP,PoPx and PM on serum HDL levels of albino wistar rats

126

4.15.4 Effect of feeding PoP,PoPx and PM on serum LDL levels of Albino wistar rats

128

4.15.5 Effect of PoP, PM and PoPx supplementation on kidney and liver function

properties of albino wistar rats ....................................................................130

4.15.6 Serum total protein contents .......................................................................144

4.15.7 Serum albumin contents ..............................................................................146

4.15.8 Serum Glucose ............................................................................................148

4.16 Rats Hematology ...................................................................................................... 151

4.16.1 Effect of feeding PoP, PoPx and PM on white blood cells of Albino wistar rats

151

4.16.2 Red Blood Cells ..........................................................................................153

4.16.3 Hemoglobin contents ..................................................................................155

4.16.4 Hematocrits .................................................................................................157

4.16.5 Platelets counts............................................................................................159

4.16.6 Lymphocytes ...............................................................................................161

4.16.7 Neutrophils counts ......................................................................................163

4.16.8 Mean Corpuscular Volume ( MCV) ...........................................................165

4.16.9 Mean Corpuscular Hemoglobin Concentration ..........................................167

5.0. Summary& Recommendations-------------------------------------------------------165

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LIST OF TABLES

No. Title Page

3.1 Standard recipe of cookies 51

3.2 Supplementation levels of PoP, PoPx and PM in cookies 51

3.3 Treatment combinations of PoP, PM, and PoPx selected for efficacy

studies 55

3.4 Standard recipe for basal diet of animal 56

3.5 Supplementation of PoP, PM, and PoPx by replacement of corn

starch 56

4.1

Extracts yield, total phenolic contents, proanthocyanidins and

antioxidant properties of PoPx at 25ºC

67

4.2

Antioxidant activity of pomegranate peel aqueous, ethanolic,

methanolic and acetone extracts

67

4.3 Urease inhibition activity of pomegranate peel extracts 70

4.4 Antimicrobial activity of standard drugs 71

4.5 Antimicrobial activity of pomegranate peel extracts 72

4.6

Minimum inhibitory concentration of PoPx for various gram

positive and gram negative microorganisms

73

4.7

Brine shrimps lethality test for pomegranate peel aqueous, ethanolic,

methanolic and acetone extracts

75

4.8

Nutritional composition of pomegranate peel, Straight grade flour

and pomegranate peel supplemented cookies (g/100g)

78

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4.9

Mineral composition of pomegranate peel and peel supplemented

cookies (mgKg-1)

80

4.10

Instrumental parameters for determination of micro elements with

atomic absorption spectroscopy

80

4.11

Total Phenolic Contents and antioxidant activity of pomegranate

peel and peel powder supplemented cookies

83

4.12 Free fatty acid (%) levels in pomegranate peel supplemented

cookies stored for a period of 4months 85

4.13 Quality scores of cookies supplemented with different levels of

pomegranate peel 88

4.14 Nutritional composition of PoPx and PM supplemented cookies

g(100g)-1 90

4.15 Total phenolics and antioxidant profile of pomegranate peel meal

and extracts supplemented cookies 92

4.16 Microbiological stability of PoPx and PM supplemented cookies 94

4.17 Thiobarbituric acid contents (mgMDA(Kg)-1) of PoPx and PM

supplemented cookies 97

4.18 Effect of PoPx and PM supplementation and storage on free fatty

acids levels of cookies (%) 98

4.19 Effect of PoPx and PM supplementation and storage on taste and

color properties of cookies 101

4.20 Effect of PoPx and PM supplementation and storage on crispiness,

texture and overall acceptability of cookies 102

4.21 Mean Square of rats’ diet intake and associated parameters 104

4.22 Effect of pomegranate peel meal supplementation on organ to body

weight (g) of albino wistar rats at different time intervals 116

4.23 Effect of pomegranate peel powder supplementation on organ to

body weight (g) of wistar rats at different time intervals 118

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4.24 Effect of pomegranate peel extracts supplementation on organ to

body weight (g) of albino wistar rats at different time intervals 120

4.25 Analysis of variance for blood chemistry 150

4.26 Effect of PoP, PM and PoPx supplementation on white blood cells

counts of albino wistar rats 152

4.27 Effect of PoP, PM and PoPx supplementation on Red blood cells

counts (millions/µl) of albino wistar rats 154

4.28 Effect of PoP, PM and PoPx supplementation on hemoglobin (g/dL)

of albino wistar rats 156

4.29 Effect of PoP, PM and PoPx supplementation on hematocrits (HCT

%) of albino wistar rats 158

4.30 Effect of PoP, PM and PoPx supplementation on platelets

(thousands) counts of albino wistar rats 160

4.31 Effect of PoP, PM and PoPx supplementation on lymphocytes (%)


4.32 Effect of PoP, PM and PoPx supplementation on neutrophils (%) of

albino wistar rats 164

4.33 Effect of PoP, PM and PoPx supplementation on mean corpuscular

volume of albino wistar rats 166

4.34 Effect of PoP, PM and PoPx supplementation on mean corpuscular

volume of albino wistar rats 168

4.35

Analysis of variance for hematological parameters of albino wistar

normal male rats feed on PoP, PM and PoPx supplemented diet for

0, 28 and 56days

169

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LIST OF FIGURES

No. Title Page

2.1 Important Ellagitannins in Pomegranate Peel & Extract 25

4.1 Effect of PoP (2.0%), PM (2.0) and PoPx (0.25%) supplementation on

feed intake 106


feed intake 106


feed intake 106


weight gain 108


weight gain 108


weight gain 108


protein intake 110


protein intake 110


protein intake 110


protein digestibility 112






feed conversion efficiency 114





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4.16 Effect of Peel Powder supplementation on serum triglycerides (mg/dL)

levels of albino wistar male rats 122

4.17 Effect of Peel Meal supplementation on serum triglycerides (mg/dL)


4.18 Effect of Peel Extract supplementation on serum triglycerides (mg/dL)


4.19 Effect of Peel Powder supplementation on serum total cholesterol

(mg/dL) levels of albino wistar male rats 125

4.20 Effect of Peel Meal supplementation on serum total cholesterol (mg/dL)


4.21 Effect of Peel Extract supplementation on serum total cholesterol


4.22 Effect of Peel Powder supplementation on High Density Lipoprotein


4.23 Effect of Peel Meal supplementation on High Density Lipoprotein


4.24 Effect of Peel Extract supplementation on High Density Lipoprotein


4.25 Effect of Peel Powder supplementation on Low Density Lipoprotein


4.26 Effect of Peel Meal supplementation on Low Density Lipoprotein


4.27 Effect of Peel Extract supplementation on Low Density Lipoprotein


4.28 Effect of Peel Powder supplementation on Uric acid function properties


4.29 Effect of Peel Meal supplementation on Uric acid function properties of


4.30 Effect of Peel Extract supplementation on Uric acid function properties


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4.31 Effect of Peel Powder supplementation on creatinine levels of albino

wistar rats 133

4.32 Effect of Peel Meal supplementation on creatinine levels of albino

wistar rats 133

4.33 Effect of Peel Extract supplementation on creatinine levels of albino

wistar rats 133

4.34 Effect of Peel Powder supplementation on Bilirubin levels of albino

wistar rats 136

4.35 Effect of Peel Meal supplementation on Bilirubin levels of albino wistar

rats 136

4.36 Effect of Peel Extract supplementation on Bilirubin levels of albino

wistar rats 136

4.37 Effect of Peel Powder supplementation on ALT (IU/L) levels of albino

wistar rats 139

4.38 Effect of Peel Meal supplementation on ALT (IU/L) levels of albino

wistar rats 139

4.39 Effect of Peel Extract supplementation on ALT (IU/L) levels of albino

wistar rats 139

4.40 Effect of Peel Powder supplementation on AST (IU/L) levels of albino

wistar rats 141

4.41 Effect of Peel Meal supplementation on AST (IU/L) levels of albino

wistar rats 141

4.42 Effect of Peel Extract supplementation on AST (IU/L) levels of albino

wistar rats 141

4.43 Effect of Peel Powder supplementation on ALP levels of albino wistar

rats 143

4.44 Effect of Peel Meal supplementation on ALP levels of albino wistar

rats 143

4.45 Effect of Peel Extract supplementation on ALP levels of albino wistar

rats 143

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4.46 Effect of Peel Powder supplementation on total protein (g/dL) levels of


4.47 Effect of Peel Meal supplementation on total protein (g/dL) levels of


4.48 Effect of Peel Extract supplementation on total protein (g/dL) levels of


4.49 Effect of Peel Powder supplementation on total albumin (g/dL) levels


4.50 Effect of Peel Meal supplementation on total albumin (g/dL) levels of


4.51 Effect of Peel Extract supplementation on total albumin (g/dL) levels of


4.52 Effect of Peel Powder supplementation on total serum glucose (mg/dL)

levels of albino wistar rats 149

4.53 Effect of Peel Meal supplementation on total serum glucose (mg/dL)


4.54 Effect of Peel Extract supplementation on total serum glucose (mg/dL)


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Chapter 1 Introduction Pomegranate (Punica granatum L) belongs to the family Punicaceae; and its common

name is derived from Latin words pomusand granatus). It is a seeded or granular apple, and

has gained tremendous popularity as a delicious fruit with increased consumption worldwide.

The fruit is native to Afghanistan, Iran, China and the Indian subcontinent. The ancient

sources of pomegranate linked Iran to Pakistan, China and Eastern India, where

pomegranates had been under cultivation for thousands of years. From the west of Persia

(modern day Iran), pomegranate cultivation stretched through the Mediterranean region to

the Turkish European borders and American Southwest, California and Mexico (Celik et al.

2009; Lansky and Newman, 2007). Pomegranate, also known in some countries as fruit of

Eden, has been praised by God (Al-Quran) for its pleasant taste and tremendous health

benefiting prospects.

Literature is not scant to confirm fruits wastes to be naturally enriched with a good

concentration of minerals, vitamins, bioactive compounds and a considerable amount of

dietary fibers. Sufficient evidence is available to designate peels and relative extracts of

numerous fruits as nutraceuticals and functional foods. Prevalence of food insecurity related

malnutrition coupled with infectious diseases warrants utilization of these healthier biological

ingredients in diets as a potential strategy to address these issues and associated health related

disorders (Akhtar et al. 2013a,b; Ismail et al. 2012). Besides being widely recognized for

bearing ethnopharmacological properties, pomegranate and its non-edible fractions such as

peel, seeds, flowers, bark, buds and leaves are also regarded as a reservoir of nutritionally

valuable components (Orgil et al. 2014). As a functional food, pomegranate fruit and fruit

extracts indicate preventive and attenuating effect against numerous health risks such as

cancer (Lansky and Newman 2007; Orgil et al. 2014), type 2 diabetes (Banihani et al. 2013)

and cardiovascular diseases (CVD) (Al-Jarallah et al. 2013). Additionally, fruit peel extract

holds exceptional free radical scavenging, anti-microbial and anti-mutational properties and

is reported to produce ameliorating effect against many critical maladies (Malviya &

Hettiarachchy 2013; Zahin et al. 2010). These health promoting features of the fruit have

lead the food entrepreneurs to focus on introducing pomegranate based food preparations

including food supplements, nutraceuticals and phenolics enriched diets (Ismail et al. 2012).

14

Bioactive fractions of pomegranate peel, particularly the minerals and fibers have

been reported to be utilized as dietary ingredients for earning associated health benefits

(Mirdehghan & Rahemi, 2007). The inorganic residues of pomegranate peel in addition to

the promising concentration of dietary fibers (12.17%) and plant phenolics (1.261%) are

reported to embrace health promoting features particularly prebiotic, anti-inflammatory,

apoptotic, hypoglycemic and anti-parasitic properties. The studies further endeavors

pomegranate peel and its other fractions to play significant role in declining rate of cardio-

vascular diseases proliferation (Abdel-Rahim et al. 2013; Anderson et al. 2009).

Pomegranate peel is characterized by the presence of fruit rind in addition to an interior

network of membranes that constitute around 26 - 30% of the entire fruit weight. The peel

fraction of pomegranate has been evidently reported to be a substantial source of a wide

range of phenolics particularly a unique class of tannins i.e. ellagitannins (ellagic acid,

punicalagin, punicalin, pedunculagin and gallic acid) and flavonoids (catechin, anthocyanins

and complex flavonoids). Ninety-two percent of the antioxidant activity reported from

pomegranate peel and the fruit juice is attributed to the phenolics mentioned above. Whereas,

punicalagin, gallic acid and ellagic acid have also been found to anticipate antimicrobial

properties against gut microflora, particularly the enteric pathogens including Salmonella

spp., E. coli, Shigella spp. and Vibrio cholera (Afaq et al. 2005; Aviram et al. 2008; Lu et al.

2007; Negi et al. 2003; Pai et al. 2011; Taguri et al. 2004; Zahin et al. 2010).

The therapeutic potential of PoP has been widely recognized by different cultures. In

Egyptian culture, several common ailments such as inflammation, diarrhea, intestinal worms,

cough and infertility have been treated by exploiting pomegranate peel extract (PoPx). The

exceptional antioxidant potential and strong medicinal properties of PoP led the international

scientific community to initiate intensive research in the last decade to further investigate its

role in human health (Lansky and Newman 2007).

Several studies have demonstrated antimicrobial, antihelminthic, and antioxidant

potential of certain active ingredients of pomegranate extracts (PoMx), suggesting their

preventive and curative role in gastro-mucosal injuries, cancer chemoprevention, ethanol-

and acetone-induced ulceration and diabetic oxidative damage (Al-zoreky and Nakahara

2003; Arun and Singh 2012; Negi et al. 2003). The mechanism of antimicrobial activity of

15

pomegranate peel phenolics involve precipitation of membrane proteins resulting in

microbial cell lysis. The ethnopharmacological profile of PoPx makes it a valuable traditional

asset due to its antimicrobial and anti-mutagenic properties. Moreover, the phytochemical

concentration of PoP is high enough to be effective without further enrichment with the

extracts of any other fraction of the fruit (Sestili et al. 2007).

Epidemiological findings on dietary consumption of phytochemicals as antioxidant

recommends plant phenolics protective role in oxidative stress. Studies consolidate oxidative

stress to be associated with emergence of certain degenerative disorders including diabetes,

cardiovascular diseases, cancer and alzheimer’s disease (Naczk and Shahidi 2006; Wong et

al. 2006). Foodborne microbiological infections and associated illnesses pose another global

challenge that distress millions of lives in multiple ways. Furthermore, emergence of drug

resistance among microorganisms of human health significance has badly impacted the

therapeutic performance of conventional antimicrobial drugs. With a viewpoint to reduce the

overwhelming and irreversible health losses associated with drug resistant microflora, some

biological compounds have shown promise as natural antimicrobials. Recent studies have

demonstrated potential therapeutic performance of some commercial forms of natural drugs

and their use has been suggested as viable strategy in combating foodborne pathogens and

associated malignancies (Techathuvanan et al. 2014).

Pomegranate, being a wonderful discovery from nature’s own resources, has been

potentially utilized for the treatment of various ailments including cancer. Data are not scant

to suggest pomegranate peel as a waste fraction to be the leading natural sources of

biological agents holding activities against foodborne pathogens (Liet al. 2014). Pomegranate

peel; constituting around 50%of the total fruit weight, is considered to be compounded with

a group of polyphenols that bear well known recognition in combating oxidative stress

associated health damages and shows broad efficacy against a wide range of microbiological

pathogenicity (Al-Saidet al. 2009; McCarrellet al. 2008; Rathiet al. 2014). Pomegranate peel

extracts obtained by using various solvents, have shown to be effective in managing

foodborne infections including Listeriosis and Candidiasis while reports are available to

recognize these extracts as effective natural sanitizers (Hayrapetyan et al. 2012; Pai et al.

2010; Tayel and El-Tras 2010).

16

Sufficient information is available to validate the chemical, biochemical and

microbiological degradation of foods and food products to be the principal causes of spoilage

which is commonly controlled with natural and synthetic antioxidants and antimicrobials

agents. On account of their food preservation properties, plant phenolics are referred as

natural and safe remedial measures in food deterioration (Maqsoodet al. 2013). Pomegranate

peel and its extracts as natural antimicrobial agents have drawn the attention of business

community to exploit them as alternative or complementary drugs in the conventional

treatment regimens (Howell and D'Souza 2013).

Pomegranate peel and whole fruit extracts have been found much effective in

controlling growth and proliferation of eleven known foodborne pathogens and their

enterotoxins (Bragaet al. 2005). Peel decoctions are generally ingested as folk medicine for

the treatment of microbial, parasitic and viral disorders including chronic diarrhea and

dysentery, and intestinal worms (Rummunet al. 2013). A wide range of activity has been

recorded for pomegranate peel extracts against foodborne pathogens, infectious zoonotic

microbiota and other spoilage factors including Staphylococcus Spp., Bacillus Spp., Listeria

monocytogenes, Yersinia eneterocolitica, Candida utilis, Aspergillu sniger, Saccharomyces

cerevisiaea and Helicobacter pylori. However, multiple drug resistance in a wide range of

microflora remains to be a distressing aspectleading to huge loss to human health and

economic conditions t (Al-Zorki 2009; Hajimahmoodi et al. 2011).

Pomegranate peel and its extracts are under application in stabilization of lipid based

food systems and to improve the physiological properties of food products. By virtue of

tremendous nutritional and nutraceutical properties of pomegranate peel and its extracts, the

fruit waste in the form of ready to use natural stabilizer and source of antioxidant have been

successfully engaged in various food preparations including meat and meat based products,

baked goods, edible oil and confectionary (Altunkaya et al. 2013a; Devatkal et al. 2012;

Iqbal et al. 2008; Kanatt et al. 2010; Naveena et al. 2008; Ventura et al. 2013). Bakery

products, specifically cookies are considered as the most viable and acceptable carriers of

such supplements (Dhingra et al. 2012). Wheat flour, being the base material of cookies is

though a good source of carbohydrates however; it may lack appreciable concentrations of

fiber, minerals and biomolecules like antioxidants to meet the growing nutritional demands

17

of the vulnerable populations. Supplementing flour for making cookies however raises

concerns with regard to consumers’ acceptability for color, taste, texture and other baking

characteristics (Kulkarni and Joshi 2013), but the product at the same time also holds

potential to fulfill nutritional needs of the body. Hard wheat-based supplemented and

fortified cookies have gained popularity among Asian populations unlike other bakery

products and are considered as specialized food groups (WFP 2013). Reliable data are

available to suggest the nutritional and nutraceutical properties of pomegranate peel and the

world’s scientific community now places a tremendous emphasis to utilize pomegranate peel

and extracts as functional ingredients and bio-preservatives in different food preparations

(Akhtar et al. 2015).

Edible part of pomegranate fruit has various applications in food industry in the form

of pure juices, concentrates, jams and jellies. An increasing trend has been observed in the

last decade for utilization of non-consumable fractions of pomegranate fruit in disease

management in terms of introducing novel functional foods. Nonetheless, consumers’

acceptance of such nutritionally altered functional foods remains to be a concern because of

their relatively reduced sensory features (Akpinar-Bayizit et al. 2012; Syedet al. 2013;

Sharma et al. 2014; Ismail et al. 2014). Little is known about the direct use of pomegranate

peel (PoP) in food recipes on account of its astringency; however, peel’s fractionated

compounds have been employed as food additives for exhibiting properties such as

antioxidant, antimicrobials, colorants and flavoring agents (Naveena et al. 2008b; Kanatt et

al. 2010; Qu et al. 2012).

Peel fraction of pomegranate is a potential reservoir of diversified polyphenols, more

specifically sugar free mono and oligomeric ellagitannins which have been frequently

utilized as natural antioxidants in various dietary supplements. Currently, commercial

formulations of pomegranate peel extracts (PoPx) based dietary supplements are available as

capsules, tablets, and soft gels. Antioxidant properties of pomegranate polyphenols have been

reported to be associated with ellagic acid, punicalin, punicalagin and gallagic acid.

Nevertheless in vivo studies suggest antioxidant properties of absorbed polyphenols to be

associated with their metabolizable compounds, e.g. urolithins (Johanningsmeier and Harris

2010).

18

Besides well-established health benefits of PoP in several ayurvedic therapies, food

use of PoP and PoPx is rare and much remains to be learnt about the possibilities of their

food applications (Ismail et al. 2012; Ismail et al. 2014). The aim of the present study is to

highlight the importance of PoP as a food bulking agent and its biological fractions as

valuable substitutes to the synthetic food additives. Moreover, the study provides baseline

information on potential food application of PoP and peel extracts (PoPx) and their

significant role in addressing the existing issues pertaining to food safety, preservation,

enrichment and quality enhancement.

The present study aims at exploring nutritional and nutraceutical potential of PoP and its

exploitation as a valuable ingredient in cereal based food products. The study is primarily an

endeavor to establish PoP as a potential source of biological antioxidants, dietary fibers and

minerals through its application in wheat flour to produce organoleptically acceptable and

nutritionally enriched cookies. Following objectives have been exclusively set to be achieved

through this research endeavor:-

Objectives:

To investigate antioxidant and antimicrobial potential of locally consumed

pomegranate peel extracts.

To evaluate lipid per-oxidation inhibitory effects of peel extract to enhance product

shelf life.

To explore nutritional potential of pomegranate peel meal (peel powder yielded after

solvent extraction) and its utilization in cookies.

To produce organoleptically acceptable peel and peel extracts based cookies and their

evaluation for antioxidant and stabilizing potential in foods.

To evaluate peel, extracts and peel meal supplemented cookies for their safety aspects

using animal models.

19

Chapter 2 Review of Literature

2.0 Preamble

This chapter comprehensively deals with the chemistry, health features and food

properties of pomegranate, the peel and other bioactive biological components of this fruit’s

edible and inedible fractions. Literature cited from the most recent updates on nutraceutical

and functional food properties of pomegranate peel and its extracts validate the waste

fraction as a broad spectrum bioactive component with a wide scope in ethnic drug

formulation and designing multipurpose nutraceutical and functional foods of choice.

2.1. Pomegranate peel phytochemistry

PoP comprises ~ 50% of fruit weight and is characterized by the presence of high

molecular weight phenolics, ellagitannins, proanthocyanidins, complex polysaccharides,

flavonoids and appreciable quantities of microelements that exhibit strong anti-mutagenic,

antioxidant, antimicrobial and apoptotic properties (Li et al. 2006; Mirdehghan and Rahemi

2007; Tezcan et al. 2009). The fruit is composed of a rich variety of flavonoids, constituting

nearly 0.2% to 1.0% of the fruit and approximately 30% of all fruit anthocyanidins are

concentrated in the peel portion (Kumari et al. 2012).

Sufficient literature confirms the presence of 124 phytochemicals in pomegranate

fruit including high molecular weight polyphenols that might exert protective effect against a

wide range of oxidative and inflammatory disorders including cancer (Heber, 2011). Nearly,

48 phenolic compounds including anthocyanins, gallotannins, hydroxycinnamic acids,

hydroxybenzoic acids and hydrolysable tannins i.e. ellagitannins, and gallagyl esters, have

been identified in PoP and other anatomical parts of the fruit (Fischer et al. 2011).

Althoughthe fruit is rich in the largest types of polyphenolic compounds including

punicalagin isomers, ellagic acid derivatives and anthocyanins (del-phinidin, cyanidin and

pelargonidin 3-glucosides and 3,5-diglucosides), PoP has been reported to contain the most

promising pool of fruit phenolics, predominately those of hydrolysable tannins as compared

to other fractions of fruit (Aviram et al. 2000; Cerda et al. 2003a; Cerda et al. 2003b; Gil et

al. 2000; Kaplan et al. 2001; Kim et al. 2002).

Mounting evidence suggests the presence of hydrolysable polyphenols in PoP, specifically

ellagitannins that render maximum free radical scavenging activity among tannins contained therein.

20

Notwithstanding, one group of investigators reviewed ellagitannin’s inhibitory activity against

oxidative changes suggesting such activities to be more likely associated with their types and

structures. Based on this concept, research endeavors were directed to explore an association

between the structures of peel phenolics and their respective inhibitory mechanism against

oxidation. These compounds [ellagic acid (hydrolyzed ellagitannins), punicalagin, punicalin,

gallagic acid] have shown to hold heightened biological activity and enhanced synergism for

each other (Seeram and Heber 2011).

High molecular weight ellagitannins are water soluble plant phenolics that yield

hexahydroxydiphenic acid on hydrolysis. Under normal physiological conditions,

gastrointestinal microflora possesses the ability to hydrolyze these orally ingested

ellagitannins to relatively smaller compound i.e. ellagic acid. Such an ellagitannin hydrolysis,

either through acid, base or gastrointestinal activity of colon micro biota, to ellagic acid has

been rampantly reported (Cerda et al. 2004; Larrosa et al. 2006; Rommel and Ronald 1993).

Punicalagin constitutes another class of health promoting larger polyphenols of

pomegranate. This compound bears good water solubility and is featured for generating

relatively smaller molecules on hydrolysis. One study demonstrated punicalagin to be the

largest ingested biomolecule in rats’ plasma besides its metabolites (Cerda et al. 2003b).

Hydrolysable tannins are reported to be the first plant polyphenols subjected to

analytical research around 200 years ago (Arapitsas, 2012); however, modern research

investigations lack to determine their functional properties and dietary role. Amongst a wide

array of PoP isolated fractions of phytochemicals, only a few have been thoroughly

investigated to date for their efficacy against certain disorders and their potential has been

technologically exploited as food additive in various dietary preparations. However, data are

scant to interpret phytochemistry, nutraceutical and food features of a substantial number of

PoP polyphenols. Albeit, major phenolics of PoP are punicalagin and its metabolites

therefore, more concerted efforts are needed to test other compounds for optimum food uses

and to establish their potential role as nutraceuticals and food additives.

21

2.2 Structure and activity relationship of PoP phenolics

Ellagitannins are commonly referred as metabolites of gallotannins. This unique

group of phenolics holds a greater tendency for hydrolysis on exposure to acidic or basic

conditions thereby releasing hexahydroxydiphenic acid (HHDP). HHDP is an unstable

group of ellagitannin that lactonizes to form a relatively stable monomeric structure of high

antioxidant potential such as ellagic acid (Kaponenet al. 2007; Aguilera-Carbo, Augur,

Prado-Barragan, Favela- Torres, & Aguilar, 2008).

PoP and mesocarp of the fruit are compounded with increased concentration of

hydrolysable tannins i.e. 27 – 172 and 32 – 263 g/Kg respectively, predominant being

monomeric phenolics (Fischer et al. 2013). Monomeric hydrolysable tannins e.g.

tellimagrandin I, strinctinin and corilagin have been reported to possess potent antibacterial

activity as compared to oligomeric tannins and di & trimericprocyanidins. Tendency of either

hydrolysable or condensed tannins or flavonoids to inhibit generation of free radicals and

associated antimicrobial features are governed by their chemical structures (Yoshida et al.

2000; Heim et al. 2002).

PoPellagitannins, bearing multiple phenolic hydroxyl groups transfer hydroxyl atom

to free radicals as free radical quenching strategy. It is one of the reasons that ellagitannins

subsequent products of oxidative reactions including dehydroellagitannins are relatively

weaker antioxidants as compared to parent compound (Okuda, 1999). A negative correlation

exists between antioxidant activity and color index of fruit as identified by Karaaslan et al.

(2014) revealing pomegranate flavonoids and phenolic acids as predominant free radical

scavengers than anthocyanins.

Ellagitannins with galloyl or hexahydroxydiphenoyl groups including casuarinin and

corilagin exhibit antiviral properties against herpes simplex virus (HSV). Monomer and

dimer ellagitannins and to some extent, gallotannins were shown to be associated with anti-

HSV-I and HSV-2 infections. Anti-infective activity of ellagitannins against foodborne

pathogens and infectious microorganisms e.g. Staphylococcus aureus, Escherichia coli, K.

pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa, and P. mirabilis have been widely

reported in the literature (Aguilera-Carbo et al. 2008; Torronen 2009; Yoshidaet al. 2009).

Exceptional ability of PoPx flavonoids to inhibit oxidative damages is ascribed to the

number and configuration of hydroxyl groups. Owing to their planarity, flavanals and

22

flavonols with 3-OH in their structure undergo conjugation and electron dislocation that

increase flavonoids phenoxyl radical stability, however; flavones e.g. luteolin lacking this

feature, are referred as weak scavenger of DPPH (Arora et al. 1998; Hirano et al. 2001).

Substitution of 3-OH group with methyl or glycosyl completely abolishes the activity of

quercetin and kaempferol against β-carotene oxidation in linoleic acid (Burda and Oleszek

2001). Presence of B-ring catechol in flavones strongly enhances lipid peroxidation

inhibition and the configuration is referred as very effective arrangement in flavonoids

against various toxic oxygen species. Luteolin and kaempferol both have identical hydroxyl

configuration however; presence of B-ring catechol in luteolin makes it superior than

kaempferol for its peroxyl radical scavenging ability (Van Acker et al. 1996). Polymerization

of flavonoids e.g. procyanidins increases the effectiveness of the compound against free

radical scavenging capacity as has been reported for dimers and trimers of procyanidins

(Vennat et al. 1994).

2.3 Traditional medicinal uses

A variety of cultures have been using pomegranate peel to treat common health

problems in both developing and developed world. Traditionally, aqueous PoP extract

generally obtained by boiling PoP for 10 - 40 min is used to treat diarrhea, dysentery, and

dental plaque, in addition to being used as a douche and enema agent (Lansky et al., 2004).

Similarly, diarrhea, intestinal worms, bleeding noses and ulcers have been treated in Indian

Subcontinent with dried PoP, plant bark and flower infusions. PoPx is gargled as a liquid to

relieve sore throat and hoarseness. Topical application of the rind powder can aid in healing

bleeding gums and plaque in patients with periodontitis (Amrutesh, 2011). Oral ingestion of

5 - 10 g of peel powder has been reportedly recommended two to three times a day for the

treatment of hyperacidity (Ismail et al. 2012).

2.4 Peel phenolics extraction modeling

Extraction of phenolic compounds from PoP is carried out by using solvents such as

methanol, ethanol, acetone, chloroform and ethyl acetate at industrial scale. Polar solvents

have greater antioxidant extraction capability as compared to the non-polar solvents.

Different solvents besides aqueous extract of the peel, were reported to yield different

phenolics content ratio and associated antioxidant activity at different extraction parameters

23

(Negi and Jayaprakasha 2003; Negi et al., 2003; Zahin et al. 2010). Phenolics extracted from

dried PoP with ethyl acetate, acetone, methanol and water revealed higher antioxidant

activity however, water extract exhibited higher anti-mutagenic activity as compared to

methanolic extracts. Due to adequate polarity of methanol, methanolic extracts of

pomegranate peel hold higher antioxidant activity comparing with other solvents (Ajaikumar

et al. 2005; Iqbal et al. 2008; Negi et al. 2003; Singh et al. 2002; Zahin et al. 2010).

Type of solvent, solid-liquid ratio, extraction temperature and size of the peel

particles have been shown to significantly affect the antioxidants extraction. Small sized peel

particles increase surface area of the powder and decrease solvent’s transfer time across the

particles subsequently yielding greater antioxidant extraction efficiency. Similarly, the total

phenolics content and antioxidant activity of PoPx increase as an inverse function of the peel

particle size. Partitioning water in methanol extracts of PoP between 2% acetic acid and ethyl

acetate increases ellagic acid yield (7.06-13.63%) and DPPH radical scavenging activity

(38.21 - 14.91 μg/mL) (Panichayupakaranant et al. 2010a). In its most economical way, PoPx

were drawn by grinding the dried peels of the fruit up to 0.2 mm mesh size and subsequently

extracted at a water/peel ratio of 50/1 (w/w) at 25°C for 2 min. Such a procedure yielded

11.5% total phenol bearing 22.9 % antioxidant content corresponding to DPPH scavenging

activity of 6.2 mgGAE/g (Qu et al. 2010).

Extraction of polyphenols with water requires higher extraction temperature, a

condition that increases the total yield but decreases the whole antioxidant activity.

Hydrolyzable tannins from PoP were extracted with the pressurized (102.1atm) de-ionized

water at 40°C up to a level of 262.7 mg/g of tannic acid equivalents. Punicalagin, the

secondary component of PoP could also be extracted up to a level of 116.6 mg/g on dry

matter basis under the same conditions (Cam and Hısıl, 2010). Pomegranate phenolics

extraction modeling has become much sophisticated in the last couple of years. Techniques

that are being applied for fractionation of active components of extracts and identification of

most appropriate extraction to raise antioxidant and anti-mutagenic activity of extracts is a

step forward to recognize the actual ethnopharmacological basis of this divine fruit.

24

2.5 Bioactivities of pomegranate peel phenolics

Antioxidant activity of PoP is associated with its phenolic compounds in the form of

anthocyanins, gallotannins, ellagitannins, gallagyl esters, hydroxybenzoic acids,

hydroxycinnamic acids and dihydroflavonol however, ellagitannins characterized by ellagic

acid, gallic acid and punicalagin are the predominant phenolics of the fruit (Cerda et al.

2003; Larrosa et al. 2006). Ellagic acid occurs in free and bound forms (EA-glycosides and

ellagitannins). Efficacy of ellagic acid as a molecular tool to treat some low to mild and

chronic disorders with very low rate of progression has been widely documented in the

literature. Moreover, it is shown to be a potential candidate as a chemo preventive agent for

cancer treatment (Kelloff et al. 1994). Beside its other well-known ethnopharmacological

properties, ellagic acid has demonstrated an ameliorating effect to reduce white fat depots

and triglycerides levels accumulated in the body during regular intake of high-fat diets.

Several studies confirmed the cytoprotective effects of ellagic acid from PoPx on oxidatively

injured living cells, oxidative DNA damage and depletion of non-protein sulfhydryl pool.

Higher ellagic acid contents are directly associated with the antioxidant activity of PoPx.

Ellagic acid contents of the fruit peel and fruit juice were reported to be 10-50 mg/100g and

1-2.38 mg/100mL, respectively (Akbarpour et al. 2009; Lu and Yuan 2008; Seeram et al.

2004).

Pomegranates have the highest concentration of punicalagins. Studies have shown

that punicalagin has antioxidant, antifungal and antibacterial properties. Alpha and beta

forms of pomegranate punicalagin are polyphenolic hydrolysable tannins and isomers of 2,3-

(S)-hexahydroxydiphenoyl-4,6-(S,S-gallagyl-D-glucose.Punicalagin, being water soluble,

hydrolyzes into smaller polyphenolic compounds in small intestine under normal

physiological conditions. Amongst peel ellagitannins, punicalagin comprises 11-20 g/kg of

the peel powder. Punicalagin available in pomegranate juice is the major antioxidant

polyphenol with an antiproliferative activity against all cell lines by inhibiting proliferation

from 30% to 100% (Fischer et al. 2011; Seeram et al. 2005).

23

Figure 2.1. Important Ellagetannins in Pomegranate Peel & Extract

25

26

2.6 Nutraceutical properties of pomegranate and peel extracts

2.6.1 Cardiovascular protective role

Atherosclerosis is one of the leading causes of death with a higher percentage in the

developed countries. LDL accumulates in the interior layers of blood vessels and then

undergoes oxidation, a process that turns them into harmful species. Inhibition of LDL

oxidation is considered to be a good strategy to prevent accumulation of foam cells and

ultimate cholesterol deposits in the arteries. Ingesting PoPx with respect to its excellent

antioxidant activity has been shown to hold potential for inhibiting LDL oxidation thereby

retarding progression of atherosclerosis with a significant reduction in arterial foam cell

levels. Pomegranate polyphenols exclusively punicalagin, gallic acid and to lesser extent

ellagic acid increase hepatocyte paraoxonase 1 expression and secretion in a dose dependent

manner thereby, reducing risk of atherosclerosis development (Khateeb et al. 2010;

Rosenblat and Aviram 2009).

Cardiovascular preventive effects of PoPellagitannins (10-100 µM) were observed in

vitro, however relatively lesser cardio protective effects of pomegranate ellagitannins were

noticed in in vivo, associating it with lesser bioavailability of fruits antioxidant fractions

(Larrosa et al., 2010). Cardio protective effects of PoMx (100 mg/kg) in rat model have been

more recently demonstrated (Hassanpour Fard et al. 2011) signifying reduction in creatine

kinase-MB, lactate dehydrogenase and glutathione. There have been many reports on the

positive effects of POM polyphenols for possessing oxidation sensitive gene and nitrous

oxide synthase expression inhibition potential, (de Nigris et al. 2005) and to reduce the

activation of redox sensitive ELK-1 and p-JUN genes and endothelial nitrous oxide

expression under induced endothelial walls shear stress (Larrosa et al. 2006; 2010). Besides

its extracts, pomegranate peel powder has also been evaluated as a dietary fiber source for the

treatment of hypercholesterolemia and atherosclerosis. Dietary supplementation of peel

powder at a concentration of 5, 10 and 15 g/100g for a period of 4 weeks significantly

reduced serum total cholesterol, triglycerides, LDL and lipid peroxidation level in

hypercholesterolemic rats (Hossin 2009).

27

2.6.2 Anti-inflammatory and anti-allergic properties

The weight of compelling scientific evidence regarding the therapeutic benefits of

pomegranate and its fractions built the scientific consensus that pomegranate rind methanolic

extract had the ability to inhibit inflammation and allergies (Panichayupakaranant et al.

2010b). Anti-inflammatory components of PoP i.e. punicalagin, punicalin, strictinin A and

granatin B significantly reduced production of nitric oxide and PGE2 by inhibiting the

expression of pro-inflammatory proteins (Lee et al. 2008; Romier et al. 2008). Evidently,

inflammatory cells including neutrophils, macrophages and monocytes may inflict damage to

nearby tissues, an event thought to be of pathogenic significance in a large number of

diseases such as emphysema, acute respiratory distress syndrome, atherosclerosis,

reperfusion injury, malignancy and rheumatoid arthritis (Babior 2000).

A recent study on isolated human neutrophils showed that aqueous PoPx directly

inhibited neutrophils’ myeloperoxidase activity, and enzyme producing hypochloric acid

from hydrogen peroxide at a concentration of 50mg/mL (Bachoual et al. 2011). One group of

investigators (Ouachrif et al. 2012) reported anti-inflammatory properties of PoPx by

interaperitoneal (25, 50 and 100 mg/kg) and intra-cerebroventricular (10, 25 and 50 µg/3

µl/rat) administration reflecting 52-82% index of pain inhibition and a significant reduction

in egg albumin induced hind paw inflammation at same intraperitoneal dosage levels.

Similarly, another study elucidated a strong inhibitory effect against inflammation

stimulators in carrageenan-induced paw edema in mice on oral administration of granatin B

(2.5 and 10 mg/kg). Significant inhibitory effects were observed after 6 hours of peel active

component administration as compared to indomethacin (Lee et al. 2010). Successful in vitro

and in vivo assays performed to evaluate anti-inflammatory properties concluded PoPx and

hydrolysable tannins in the form of standardized active components, to be very effective

remedial measure against inflammatory disorders.

2.6.3 Anticancer perspectives

Cancer is the leading cause of death both in the developed and developing countries.

The menace is prevalent in low and middle income population with higher mortality rates.

The significant determinants for increased death rate in poor economies accounts for a

complete absence of appropriate health systems. Estimates showed 41% increase in cancer

28

cases from 1975 to 2008 and the proportion seems like escalating to 70% by 2030. The early

detection and practicing appropriate preventive measures as strategies to reduce the cancer

burden have been widely recommended by health professionals (Ferlay et al. 2010; Kanavos

2006; Sloan and Gelband 2007).

ROS are known to represent a causal and/or concausal factor in the development of

cancer. Extensive damage to DNA by ROS ultimately leads towards somatic mutations and

organ malignancies. ROS produced during normal cellular metabolic processes, or derived

from the exposure to ionizing radiations or xenobiotics are well recognized concausal factors

in a wide number of chronic diseases, including CVD and cancer. ROS toxic effects depend

on their capacity of damaging relevant and sensitive biological substrates, such as DNA,

RNA, proteins and membrane lipids. ROS include superoxide radicals, lipoperoxides,

hydrogen peroxide, hydroxyl free radicals (Wiseman and Halliwell 1996).

Indiscrete chemotherapy to ameliorate oxidative damages in human has not been

regarded as comforting therapeutic tool to treat malignancies. At cellular and tissue level,

copper and iron binding sites of macromolecules serve as central sites for free radicals

production. Such site specific free radicals generation is inhibited by chelation of metal ions

by antioxidants of biological origin e.g. flavonoids (Chevion, 1988; Sies, 1997). Moreover,

ellagic acid and punicalagins arrest cancer cell growth by inducing apoptosis-a multistep cell

death program. Antioxidants, either from synthetic or plant sources, are thought to be a

preventive approach against carcinogenesis. Natural antioxidants, despite the presence of

synthetic ones in the market, gained a wider acceptance in relation to their safe edible limits.

Prostate and Colon cancer

The anticarcinogenic effects of the fruit ellagitannins were associated with its hydrolyzed

products specifically ellagic acid and punicalagin that induced apoptosis in colon cancer cells

(HT-29, HCT116) and prostate cancer cells at a concentration of 100 μg/mL through some

fundamental pathways like introducing cytchrome “c” in cell cytosol and by up-regulation of

Bax and down-modulation Bcl-2 (Larrosa et al. 2006; Malik et al. 2005; Seeram et al. 2004;

2005). Treatment of Los Angeles prostate cancer cells (LAPC4) with standardized POMx (10

µg/mL) containing 37% punicalagin have strong roots in inhibition of cell proliferation and

29

apoptosis induction. PoMx treatment in combination with IGFBP-3 reduced the activity of

cell growth promoters (Akt and mTOR) performing a pro-apoptotic function in inhibiting

cancer cells growth (Koyama et al. 2010).

In vitro incubation of human prostate cancer cells and umbilical vein endothelial cells

with 37% standardized ellagitannins-rich PoMx inhibited proliferation of both prostate

cancer and umbilical vein endothelial cells under hypoxic and normoxic conditions. IC50 of

ellagitannin enriched PoMx for endothelial cells proliferation were recorded as 6.7 ± 0.5

µg/mL and 2.2 ± 0.2 µg/mL under reduced and normal oxygen supply, respectively. Similar

inhibitory effects were observed by oral administration of ellagitannins-rich PoMx for a

period of 4 weeks to mice subcutaneously injected with prostate cancer cells. Indeed, a

momentous reduction in cancer cells proliferation, prostate cancer xenograft size and tumor

vessel density were observed (Sartippour et al. 2008). Koyama et al. (2010) demonstrated

complete inhibition of PoMx induced apoptosis on a 100 ng/mL co-treatment of prostate

cancer cells with IGF-1. However, much remains to be learnt about positive and negative

synergistic effects of PoMx with cell growth factors.

2.6.3.1 Melanogenesis/ Skin cancer

PoPx (IC50=182.2 µg/mL) inhibited melanocytes proliferation and melanin synthesis

by inhibiting tyrosinase activity. The magnitude of inhibition wasmore likely comparable

with arbutin. Protective role of orally administrated PoPx containing 90% ellagic acid at the

rate of 1000mg/kg as compared to water fed group have been demonstrated in brown guinea

pigs to inhibit skin pigmentation induced by exposure to UV radiations (Yoshimura et al.

2005).

Several studies confirmed the ability of PoPx and PoM ellagitannins (500 –

10000mg/L) to inhibit free radicals generation in UVA and UVB irradiated human skin thus

protecting it from DNA fragmentation, skin burns and depigmentation. This human skin

DNA base damage is attributable to monochromatic light that activates photosensitizers

leading to the generation of genotoxic single oxygen species (Kasai et al. 2006; Manasathien

et al. 2011; Pacheco-Palencia et al. 2008). Another study explicated the role of PoMx in

mediating UVB induced skin damage. Epidermal pretreatment of POMx (5–10 μg/0.1

mL/well) to UVB (60 mJ/cm2) induced skin damage inhibited the matrix metalloproteinases

30

compounds involved in degradation of skin connective tissues and collagen components and

the markers of oxidative stress and genotoxicity (Afaq et al. 2009).

2.6.3.2 Breast Cancer

PoPx have shown to induce apoptosis in human breast cancer cells (MCF-7).

Therapeutic application of PoPx with genistein illustrated significantly higher MCF-7

inhibitory and cytotoxic effects in the treatment of breast cancer. Moreover, PoPx exhibited a

potential to inhibit the expression of markers of cell proliferation and angiogenesis,

phosphorylation of p38 and C-Jun mitogen-activated protein kinases and activation of pro

survival signaling pathways. PoPx have further shown to inhibit nuclear factor kappa B (NF-

kB)-dependent reporter gene expression associated with proliferation, invasion, and motility

in aggressive breast cancer phenotypes (Jeune et al.2005; Khan et al. 2009).

Quite a number of reports presented reasonable explanation of endocrinal therapy of

breast cancer in post-menopausal women where non- significant response to estrogen

receptors-positive MCF-7 breast tumors was observed. PoMx exhibiting anticancer

properties were successfully tested at a rate of 300 μg/mL in combination with 1μM

tamoxifen to sensitize and enhance latter activity thereby inhibiting resistant MCF-7 cells

proliferation (Aiyer et al. 2012; Banerjee et al. 2011).

Breast cancer cells proliferation and generation of estrogen receptor-positive tumors

are estrogen stimulated activities that could be checked by anti-aromatase compounds.

Urolithin B from the metabolites of pomegranate ellagitannins has been identified by

microsomal aromatase assay as the active ingredients with maximum antiaromatase activity

beside inhibition of testosterone-induced MCF7 cells proliferation (Adams et al. 2010).

Although in vitro cultured cell and animal anti-estrogenic studies on pomegranate extracts

constituents have been successfully executed however; human studies are required to clarify

the effect of PoMx and PoPx as nutraceutical on serum hormone levels and associated

activity in a more systematic way.

31

2.6.4 Antimicrobial potential of peel extracts

Polyphenols, flavonoids, condensed and hydrolysable tannins extracted from fruits,

vegetables, herbs and spices have been explored as potential agents for treating or preventing

a wide range of infective disorders (Cowan, 1999; Naz et al. 2007; Taguri et al. 2004).

PoPx are conventionally recommended as an ethnopharmacological remedial treatment of

foodborne diseases and urinary tract infections in Indian subcontinent (El-Sherbini et al.

2010;Gopalakrishnan and Benny 2009). The antimicrobial mechanisms of phenolic

compounds involved, reaction of phenolics with microbial cell membrane proteins and/or

protein sulfhydryl group yielding bacterial death due to membrane protein precipitation and

inhibition of enzymes such as glycosyltransferases (Haslam 1996; Naz et al. 2007;

Vasconcelos et al. 2003).

Evidently, PoPx has shown promising results as an agent to treat food borne pathogenicity

and urinary tract infections (El-Sherbini et al. 2010; Gopalakrishnan and Benny 2009), while

PoP ellagitannins, punicalagin, ellagic acid and gallic acid, as natural antimicrobial agents,

have been widely exploited against Staphylococcus aureus and hemorrhagic Escherichia

coli for their ability to precipitate membrane protein and inhibition of enzymes such as

glycosyltransferases that lead to cell lysis (Braga et al. 2005; Haslam, 1996; Machado et al.

2003; Naz et al. 2007; Vasconcelos et al. 2003; Voravuthikunchai et al. 2005).

In vivo and in situ application of 80% methanolic extract of PoP revealed its inhibitory effect

against Listeria monocytogens, Staphylococcus aureus, Escherichia coli and Yersinia

enterocolitica (Al-Zoreky, 2009). However, higher doses of PoPx (24.7 mg/mL) were

reported to be the minimum bactericidal concentration for Listeria monocytogenes.

2.6.4.1 Microbial inhibitory mechanism of peel extracts

Plant phenolics have the ability to generate complexes with microbial cell membranes

and its soluble proteins. Anti-peroxidation features of tannins (hydrolysable and condensed)

are reported to inhibit microbial growth particularly E. coli (Cowan 1999; Fowler et al. 2011;

Okuda 2005; Cimolai and Cimolai 2007). Numbers of phytochemicals are associated with

antimicrobial activity of pomegranate peel extracts but ellagic acid and punicalagin excel

32

others for the same (Howell and D’Souza 2013). Antimicrobial property of pomegranate or

its peel extracts is greatly associated with tannins predominately punicalagin and ellagic acid

that account for 64.2% and 3.1% of rind extracts, respectively (Li et al. 2014). Studies

suggest reduction in intracellular ATP concentration and contrarily, its higher concentration

with that of potassium outside the cell as the factors responsible for PoPx induced

microbiological inhibition. Seemingly, pomegranate phytochemicals impair integrity of the

cell membranes by hyperpolarization and reduction of pH inside the cell consequently

resulting in loss of cell homeostasis (Li et al. 2014).

2.6.5 Anti-influenza and anti-malarial responses

Pomegranate hydrolysable tannins including punicalin, punicalagins, gallagic acid

and ellagic acid exhibited antiviral properties to modulate respiratory infections and

influenza (Gil et al., 2000; Nonaka et al., 1990). Mechanistic inhibition of RNA replication

of the influenza virus by pomegranate purified polyphenol extract is attributed for such

antiviral properties. Punicalagins with inhibitory concentration of up to 40 µg/mL have

shown to be the most active in blocking the replication of the virus RNA (Haidari et al.

2009). Similarly, peel phenolics viral inactivation mechanism involved influenza virus direct

structural damages and indirect intercellular inhibition of viral replication. Viruses enveloped

with glycoprotein were reported to be more susceptible to structural damages by polyphenols

as compared to non-enveloped ones (Kotwal, 2008).

Another recent study elucidated antiviral potential of pomegranate polyphenols

reporting a short time (5min) exposure of avian and human influenza viruses (H1N1, H3N2,

and H5N1) to 800 µg/mL pomegranate polyphenols that revealed 3 log reduction of viruses

at normal temperature (Sundararajan et al. 2010). However, antiviral activity of the

polyphenols was less effective against H5N1 influenza virus isolated from the avian.

Virucidal effects of pomegranate phenolics are associated with interaction of pomegranate

phenolics with viral neuraminidase yielding loss of red blood cells agglutination.

Pomegranate phenolics interact at the intercellular steps and inhibit the replication of

influenza virus as well (Haidari et al. 2009).

OMARIA, an ayurvedic formulation derived from the dried PoP powder is a basic

nutraceutical being used for the treatment of malaria in the Plasmodium falciparum and

33

Plasmodium vivax endemic rural area of the eastern province of Orissa, India. Interestingly,

chloroquine resistant strains of Plasmodium falciparum were found to be sensitive to the

toxic action of methanolic extracts of PoP (Dell’Agli et al. 2010).

Tannins enriched POM methanolic extract tested against haemozoin (6μg/mL)

stimulated THP-1 cells antagonistically reduced MMP-9 secretion by 78 and 95% at

concentration of 50 and 100 μg/mL, respectively. Nearly, 65 and 79% inhibition of MMP-9

secretion in THP-1 stimulated cells were noticed at 10 μM concentration of the purified

component of extracts i.e. ellagic acid and punicalagin, respectively (Dell’Agli et al. 2010).

2.6.6 Wound healing potential

Epithelialization, antioxidant immunity and characteristic biochemical properties are

the common features of wound healing process that predominantly prevails in injured tissues.

Topical administration of PoPx can be recommended for dead space, incisional and

excisional wound models. Improved epithelialization, breaking strength and contraction of

incised wounds, along with increased hydroxyproline content, dry weight and breaking

strength of granulated tissues, can be observed in the healing process of wounds treated with

PoPx. Oral administration of 100 mg/kg aqueous extract of PoPx to wistar rats and external

application of PoPx formulated with hydrophilic gel reported significant improvement in all

wound models (Adiga et al. 2010; Murthy et al. 2004).

Methanolic extracts of PoP exhibited a potent inhibitory effect in gastric mucosal

injuries. Oral administration of 70% methanolic extract of peel at a rate of 250 mg/kg and

500 mg/kg inhibited the progression of ulcers by 22.37, 74.21 and 21.95, 63.41% in rats with

aspirin and ethanol induced gastric ulcers, respectively (Ajaikumar et al. 2005).

2.6.7 PoPx and oral pathogens

Natural products are effective adjuvant to the pharmaceuticals revealing their

judicious use to not only encounter microbial infections but also to cut down side effects of

synthetic drugs at relatively lower cost. It is one of the reasons that developing economies

have higher rate of reliability on ethnic natural medicines as compared to pharmaceutics.

Pomegranate peel extracts may be brought in practice to protect oral health from microbial

pathogens responsible for periodontal diseases, dental caries and stomatitis. Methanolic

extracts of pomegranate peel were found effective against Staphylococcus aureus and S.

34

epidermidis. Relatively higher concentrations of extracts i.e. 8 and 10mg/L were found

effective in inhibiting some oral pathogens including L. acidophilus, S. salivarius and S.

mutans (Abdollahzadeh et al. 2011).

Fermentation of carbohydrates caught between the teeth helps produce gingivitis

characterized by the bleeding and swelling of gums. Pomegranate peel based extracts prevent

formation of dental plaque whereas the compound believed to be the remedy, were found to

be the flavonoids and peel phenolic acids. Mouth rinsing with pomegranate peels extracts

affects multiple oral health indicators including reduction of total protein, reduction in

activities of enzymes responsible for sucrose degrading and protection of cell injury

(DiSilvestro et al. 2009). Hydroalcoholic extracts of pomegranate greatly inhibit the growth

of oral microflora responsible for dental plaque. Mouth rinsing with pomegranate

hydroalcoholic extracts inhibit 84% of oral microflora however; in vivo studies reveal 5%

lower rate of total plate count elimination vide application of standard mouth wash

ingredients i.e. chlorhexidine (Menezes et al. 2006). Pomegranate juice without sugar has

even been reported to inhibit Streptococci and Lactobacilli from oral cavity by 23 and 46%

respectively (Sowmya Kote et al. 2011) that further authenticate pomegranate and its

anatomical fractions as remarkable biological element to protect oral health.

2.7 PoP and PoPx - a biological class of food additives

Utilization of functional foods as preventive therapeutic approach against digestive

disorders, cardiovascular risks and various types of cancer has been widely recognized

(Akhtar et al. 2013b). Consumption and marketing of pomegranate and its products have

been rapidly expanding worldwide on account of their functional properties and potential to

prevent disease. Moreover, fruit utilization as food colorants and flavor enhancers, has also

been reported in the literature (Al-Maiman and Ahmad, 2002).

Pomegranate processing industries produce huge waste in the form of peel that is

normally utilized as animal feed. Data are scant to elucidate nutritional composition and food

features of PoP; however, tremendous research has been directed to explore their antioxidant

contents and possible anti-inflammatory and anti-infective role against a wide array of

infections and mild to chronic health disorders (Ismail et al. 2012). Astringency is the key

35

limiting factor in its utilization as food despite its outstanding nutritional and

ethnopharmacological significance. Bitter sensation in PoP and juice primarily emanates

from combination of low and high molecular weight flavanoids and tannins with

carbohydrates forming complex compounds (Hollman and Arts 2000; Martin and Appel

2010).

Inadequate supply of certain vital nutrients in regular dietary plan has been reported

as a corollary of increased malnutrition associated morbidity and to some extent maternal as

well as infant mortality (Akhtar et al. 2013a). PoP has significant concentration of macro and

micro nutrients that varies among cultivars and regions. Several studies confirmed peel

powder to be rich in minerals, complex polysaccharides, reducing sugars, fiber and protein,

suggesting its utilization as a valuable ingredient in foods (Singh et al. 2007; Viuda-Martos

et al. 2010).

A group of researchers demonstrated exceptional nutritional composition of PoP

(Ramadan et al. 2010) suggesting peel powder to be used as a cherished raw material for the

preparation of secondary food products i.e. food colorant (β-carotene up to 13.67mg/kg) and

nutrient rich formulas containing Na, K, Ca, Fe, Mn, Mg, ascorbic acid and total sugars

(Singh et al. 2007; Ullah et al. 2012; Viuda-Martos et al. 2012; Viuda-Martos et al. 2013).

Moreover, on account of its mineral profile, pomegranate harvested in early season has also

been reported to contain higher calcium and sodium contents as compared to the late crop

(Marschner, 1995; Mirdehghan and Rahemi 2007).

2.7.1 Antioxidants potential of PoP and PoPx

Oxidation is a fundamental deteriorative change in foods containing lipid fraction

during processing and subsequent storage conditions. Visible onset of lipid oxidation is

believed to result in negative nutritional and sensorial alteration of foods (Shahidi et al.

1992). Plant phenolics and derived compounds have been extensively studied for their

mechanistic autoxidation reduction and preventive role. There has been a plausible

explanation in the literature to validate natural plant extracts to be less toxic and more

effective as compared to synthetic antioxidants against certain health disorders and food

spoilage mechanisms. Presence of natural ingredients such as antioxidants in food products

enhances consumer acceptability in addition to improving stability of the products (Al-

36

Zoreky and Nakahara 2001; Han et al. 2008; Negi and Jayaprakasha 2003; Reddy et al.

2007).

Several studies elucidated natural sources, such as plants, to be rich in non-toxic

biologically-active compounds like polyphenols. Synthetic antioxidants have been

industrially used as food additives for more than fifty years as a means to prevent

peroxidation of fats and oils. Butylated hydroxyl toluene (BHT), Butylated hydroxyl anisole

(BHA) and tert-butylhydroquinone are effective and common antioxidants preventing

oxidation and off-flavor development in fats and oils. However, safety of these compounds

remains a question as numerous studies exhibited their damaging effect on health suggesting

their restricted use in food products (Han et al. 2008; Iqbal et al. 2008).

PoPx, evaluated by FRAP assay was found to be the richest source of antioxidants

amongst peel extracts. Similarly, PoP demonstrated 2.8 times higher antioxidant activity as

compared to pomegranate seed and leaf extracts (Guo et al. 2003; Okonogi et al. 2007; Negi

et al. 2003; Tehranifar et al. 2011).Studies have provided a deep insight into our

understanding of the nutritional and nutraceutical potential of PoP suggesting peel and PoPx

to be used as a functional food ingredient and a bio-preservative in different food

applications. To this regard, one most recent study on wheat bread fortified with PoP powder

@ 10% confirmed increased oxidative stability with least peroxide contents of product.

However, higher concentrations of peel were also observed with higher rate of brine shrimps

larval death thereby indicating possible toxicological impact of peel powder at

supplementation levels higher than 2.5% (Altunkaya et al. 2013a). One group of researchers

reported PoP methanolic extracts at a concentration of 1000ppm to act as potent antioxidants

in sunflower oil at accelerated processing conditions, i.e. higher temperature (185˚C) and

prolonged heating duration (80 min) (Iqbal et al. 2008).

2.7.2 PoP and PoPx as dietary supplements

Evidently, dietary polyphenols utilization under permissible limits may generate

beneficial health effects (Hu, 2007). Restricted daily consumption of fruits and vegetables by

populations of developed and resource less countries results in decreased dietary intake of

plant polyphenols. Pomegranate extracts, either from juice or peels, have shown promise as a

rich source of phytochemical and associated free radical scavenging activity. Relatively

37

higher proportion of polyphenols-natural antioxidants exists in peel as compared to juice,

seed and flower fractions of fruit (Li et al. 2006). Production of potentially health beneficial

dietary supplements as by-product of pomegranate juice processing industry waste has shown

to heighten the economic growth of food industry. This notion seems to be true as dietary

supplements have been formulated in the form of gels and capsules; however there still exists

a growing demand for more specific and purified fractions of PoPx as a more specific

treatment regimen.

2.7.3 Stabilization of unsaturated fatty acids in food systems

Oxidation is a fundamental deteriorative change in foods containing lipid fraction

during processing and subsequent storage conditions. Visible onset of lipid oxidation is

believed to result in negative nutritional and sensorial alteration of foods. Synthetic

antioxidants have been industrially used as food additives for more than fifty years as means

to prevent peroxidation of fats and oils. BHT, BHA and tert-butylhydroquinone are effective

and common antioxidants preventing oxidation and off-flavor development in fats and oils.

However, safety of these compounds remains a question as numerous studies exhibited their

damaging effect on health suggesting their restricted use in food products (Iqbal et al. 2008;

Ahmad et al. 2013).

Some of the synthetic antioxidants have been found to cause mutation in normal cells.

It’s one of the reasons that utilization of synthetic antioxidants in food systems has

perpetually declined since last decade on account of their deleterious effects on health at

prolonged exposures. However, natural antioxidants including ascorbic acid, tocopherols,

and plant phenolics rich extracts have gained significant recognition worldwide (Frost and

Sullivan 2013). PoPx could be used as natural antioxidant for the stabilization of vegetable

oils thereby substituting synthetic antioxidants. e. g. BHT and BHA (Iqbal et al. 2008).

Several foods undergo deteriorative changes during preparation and storage on

account of lipid oxidation leading to the development of off-flavors and production of certain

toxic compounds. Predominant precursors engendering these qualitative alterations in such

foods include enzymes, metal ions, ionizing radiations and sunlight. Utilization of PoP

powder extracts has been reported to stabilize the food systems against such lipid oxidative

changes. Significantly higher phenolics profile of PoP characterized by hydrolysable tannins

38

i.e. ellagitannins, ellagic acid, punicalin and punicalagin isomers have shown to possess

increased potential to inhibit lipid oxidation and free radicals scavenging properties (Li et al.

2006). Several studies demonstrated remarkable effects of PoPx on the product stability

against lipid peroxidation of vegetable oil, cooked chicken, beef and goat meat patties (Iqbal

et al. 2008; Kanatt et al. 2010; Naveena et al. 2008a;Naveena et al. 2008b).

Stability of the phenolics rich extracts and individual bioactive compounds at

processing conditions can potentially compromise their nutritional and nutraceutical efficacy.

PoP liquid extracts manifest reasonable stability at chilling temperature and are thus referred

as useful natural additives in various perishable food commodities either processed or stored

at chilling or refrigeration temperatures. Six-month storage studies reports punicalagin A and

B, gallic and ellagic acid enriched natural extracts of PoP to have better stability (67%) and

free radical scavenging properties (58%) at 4˚C (Qu et al. 2013).

PoPx rendered enhanced antioxidant activity by inhibiting lipid oxidation as

compared to many natural and synthetic antioxidants such as Vitamin C and BHT in cooked

chicken patties (Naveena et al. 2008a;Naveena et al. 2008b). Similarly, presence of higher

concentration of TBARS (Thiobarbituric acid reactive substances) is an indicative of

oxidative damage in meat and meat products. PoPx in combination with vacuum packaging

technology has been evaluated for its effect on reduction of TBARS and to preserve

organoleptic attributes of the product. Supplementing 1% PoPx in goat meat reduced TBARS

by 40% as compared to individual vacuum packaging where the reduction rates remained to

be 27% (Devatkal et al. 2012).

2.7.4 PoP and PoPx as barriers to food spoilage and infections

A notion prevails in the world’s scientific community regarding emergence of multi-

drug resistance food borne pathogen strains e.g. Staphylococcus aureus, Salmonella

enteritidis and Listeria monocytogens that are recognized as potential threats to safe food

supply leading to higher rate of morbidity and mortality (Akhtar, 2015; Akhtar et al. 2014).

More recently, astrong affinity of PoPx toward inhibition of gram positive foodborne

pathogens in ready to eat meat preparations was reported, suggesting its utilization as a

potential food safety approach in meat and meat products (Hayrapetyan et al. 2012).

39

In vivo and in situ application of 80% methanolic extract of PoP revealed a potential

inhibitory effect for L. monocytogens, S. aureus, E. coli and Y. enterocolitica. Minimum

inhibitory concentration (MIC) of the water methanolic extract as 4mg/mL for Salmonella

enteritidis while 24.7mg dry Px/mL was reported to be the minimum bactericidal

concentration for L. monocytogenes (Al-Zoreky, 2009; Hayrapetyan et al. 2012).

Abundant literature is available to confirm antibacterial activity of pomegranate

polyphenols that are hydrophilic in nature and are well extracted with the help of hydrophilic

extractants. Ellagitannins isolated from PoP have been reported to exhibit antibacterial

activity against both methicillin-resistant and methicillin-sensitive S. aureus, with MICs of

62.5 µg/mL (Negi and Jayaprakasha 2003; Naz et al. 2007). Contrarily, hydrophobic

extractants i.e. ethyl acetate, chloroform and n-hexane were reported to carry

microbiologically inactive compounds in their extracts and did not yield any antimicrobial

activity (Al-Zoreky and Nakahara 2003).

Mechanistically, precipitation of bacterial cell membrane proteins by the reaction of

peel phenolics entails bacterial cell lysis. Likewise, phenolic compounds may react with

protein sulfhydryl group making them unavailable for microbial growth thereby generating

phenolics toxicity (Haslan, 1996; Negi and Jayaprakasha 2003).

2.7.5 PoP enhances functional quality of foods

Addition of pomegranate rind powder in raw beef sausages up to 3% has been

reported to improve their functional characteristics i.e. water holding capacity of sausages in

addition to higher phenolics and free radical scavenging activity. Similarly, supplementing

(3%) pomegranate rind powder characteristically improved the quality (hue, chroma,

lightness and redness) of cooked meat sausages suggesting whole fruit bagasse as a potential

food ingredient with functional food properties (El-Gharably and Ashoush 2011).

Another recent study confirmed nutritional significance of pomegranate bagasse

showing it to be a potential source of dietary fiber i.e. total, soluble and insoluble dietary

fiber 50.3, 19.9 and 30.4g/100g, respectively. The study further explicates pomegranate

bagasse powder co-products to be exploited in food products requiring hydration, viscosity

development, and freshness, such as baked foods or cooked meat products (Viuda-Martos et

al. 2012). Based on the data available on functional characteristics of PoP powder and

40

extracts, more focused studies are needed on regional cultivars grown in a diversified

climacteric conditions.

2.7.6 PoP and PoPx as prebiotics

Prebiotics, food ingredients with no digestibility, hold a strong affinity towards

improving selected or randomized growth of colon microbiota. PoPx carrying appreciable

concentration of ellagitannins are hydrolyzed by intestinal microflora into punicalagin and

ellagic acid. Individually fractionated hydrolyzed products of ellagitannins reportedly form

dibenzopiranonas, i.e. urolithins A and B; however, colon microbiota stand responsible for

these actively metabolized by-productsis still unexplained (Gibson et al. 2004). More recent

studies are available to interpret the mechanistic role of prebiotics to inhibit pathogens and

promote the growth of beneficial micobiota by competing for adhesion sites and nutrition

(Collado et al. 2009; Quigley 2010). Pomegranate extracts, in the same way, exhibit prebiotic

activity for gastrointestinal microbiota by inhibiting growth of pathogenic microorganisms

(Collins and Gibson 1999).

Prebiotic activity of PoPx has been extensively evaluated against inflammatory and

hypercholestrolemic disorders (Neyrinck et al. 2012). The system involving interaction of

ellagitannins, accumulated in large intestine, with gut microbiota suggested synergism

between ellagitannins and gut microbiota as influenced by tannin and specie type.

Consumption of pomegranate extracts selectively promotes growth of Bifido

bacteriuminfantis and B. breve and inhibits growth of pathogens including S.aureus and

Clostridia (Bialonska et al. 2009).

41

2.8 Functional and toxicological levels of PoP and PoPx for food uses

PoPx like other biological compounds might generate toxicity if consumed or

exposed at levels more than threshold limits. Lethal doses or concentration of PoPx and some

fractionated compounds have been worked out in vitro and in vivo in the last couple of years.

Considerable progress has been observed in toxicity monitoring and establishing no observed

adverse effect levels of PoPx after the work of Vidal et al. (2003). Upper levels of PoMx,

PoPx and fractionated compounds (>2000mg/kg b.w) were evaluated for suspected toxicity

in laboratory animals (Vidal et al. 2003).

Galactomannan polysaccharides exhibiting cytotoxic properties against cancer cells

were isolated from PoP. In vivo administration of galactomannan polysaccharides to BALB/c

mice at a concentration of 2000mg/kg b.w. did not reflect any toxic effect revealing LD50 of

the tested compound to be higher than 2000mg/kg b.w (Joseph et al. 2013). Parallel findings

have also been reported for synthetic ellagic acid and PoMx (Bhandary et al. 2013). Most

recently oral administration of PoM ethanolic extracts to female rats at a concentration of

2000g/kg b.w. did not present any toxicity in the tested animals (Das and Sarma 2014).

Higher level of variability in toxicological concentration of PoPx administrated

intraperitoneally in rats and mice has been reported probably due to variability in

composition of peel biological extracts. PoPx subjected for antidiarrheal activity screening

were intraperitoneally administrated to rats and established 1321mg/kg b.w. of the injected

therapeutic as LD50 (Qnais et al., 2007). Later on comparatively 100% lower LD50 for acute

and sub-chronic intraperitoneal administration of PoPx in wistar rats were also reported by

Patel et al. (2008). Being most convenient to work on, probable toxicological studies of POM

and PoPx has been performed on brine shrimps (Artemia salina). Lethal concentration of

methanolic extracts of PoP to inhibit 50% population of Artemia salina screened against

enteric pathogens was found as 1.42mg/mL (Mehru et al. 2008).

Safe and intelligent utilization of PoP and its extracts in food products for the sake of

extended nutraceutical and functional features are underway. In the same context, PoP

supplementation in bread at a level of 2.5% were found safe however; concentration >2.5%

were found to significantly raise death rate of Artemia salina (Altunkaya et al. 2013a). The

42

same organism has also been tested for toxicity from PoPx enriched apple juice and doses

lower than 1000mg per 100mL were referred as safe (Altunkaya et al. 2013b). Previously,

food application of PoPx up to a level of 1420mg were suggested as safe revealing no

significant histopathological effects in normal and obese individuals (Heber et al. 2007).

2.9 Limitations and future directions

Although a remarkable progress has been observed in the last couple of years in

chemical characterization of pomegranate phytochemicals, development of extraction kinetic

models with variant process parameters, exploring therapeutic potential and molecular targets

but still there are some avenues that need to be investigated before suggesting future

recommendations.

Reduction in therapeutic outcomes of PoMx on persistence application has emerged

as a concern for the treatment of chronic oxidative injuries. Comprehensive and long term

therapeutic studies must be carried out against chemically induced or chronic oxidative

injuries. Traditional utilization of the PoMx, PoPx and the fruit juice for the treatment of

diarrheal disorders has been well explained in terms of fruit extracts antimicrobial potential

against potential pathogens. Some more emerging issues like candidiasis demand in vivo

trials to draw a clearer picture of the fruit potential for the treatment of fatal in vivo fungal

proliferation. Once identified, the extracts should be evaluated for their prophylactic potential

against viral epidemics and pandemics specifically influenza. Fate of urolithins and its

conjugated forms concentration and bioavailability in plasma is still unclear that need to be

addressed. Toxicity studies are confined to the higher doses of pomegranate phenolics

however a sizeable review of literature reveals no report on isolation and quantification of

toxicological components of the fruit and fruit extracts. Relative toxicity of PoMx, PoPx and

individual purified phytochemicals carrying therapeutically significant health beneficiary

effects must be evaluated before potential application.

Pomegranate is ranked as a fruit that manifests highest antioxidant potential while its

peel fraction has been acknowledged as bearing the highest antioxidant potential

phytochemicals pool. A plethora of literature is available to highlight ethnopharmacological

and nutraceuticalfeatures of pomegranate and its peel fractions in addition to their potential

to act as health ameliorating biological ingredients. Numerous in vitro and in vivo studies

43

have validated exceptional antioxidant potential of the peel and its curative properties against

some critical diseases like prostrate, colon and liver cancers, stomach ulcers, cardiovascular

diseases and digestive disorders revealing their potential cytoprotective and inhibitory

effects.

PoMx and PoPx could serve as good preventive strategy against some human

carcinomas and, to some extent, a therapeutic agent enhancing the life span. A lot of work

has been published and a lot more is in progress on ethnopharmacological utilization of fruit

and peel extracts. Preventive and therapeutic potential of extracts and standardized bioactive

compounds of pomegranate has been established however, validation still demands critical

appraisal on safe and practical application of this phytonutrient pool in human.

There have been fewer reports on their possible toxicology, dietary ranges and

consumption patterns. Utilization of pomegranate peel and its extract in defined

concentrations in various organoleptically acceptable food preparations, as effective

supplements and food additives, would open new avenues for scientific research in the realm

of food science and nutrition. Incorporation of PoP or its fractionated phytochemicals

regardless of their astringency could practically be exploited for their health promoting

features in various food products with slight but acceptable sensorial modifications.

Exploitation of pomegranate peel as a reservoir of valuable therapeutic agents that may also

act as food preservatives, stabilizers, supplements, probiotics and quality enhancement

agents seems to be a pragmatic disease preventive approach, however, careful exploration to

spotlight the efficacy of pomegranate peel and peel extract and its nutraceutical role as

supplements in food, stability of active ingredients under various food processing conditions

and sensory defects in finished food products, is needed to fully exploit the waste of this

heavenly fruit.

44

Chapter 3 Materials & Methods 3.1 Procurement of Raw Material

Local white cultivar of pomegranate “Ali Puri” characterized by yellow rind, white juicy

sweet sacs with evenly distributed partially red grains was procured from a farm of Alipur,

29° 23' 0" North and 70° 55' 0" East of District Muzaffar Garh,Punjab- Pakistan. Fruit

samples so obtained were got verified for the variety from fruit plant experts at Faculty of

Agricultural Sciences and Technology, Bahauddin Zakariya Multan, Pakistan. A reference

specimen from the sampled population was kept for record in the Institute of Food Science &

Nutrition

3.2 Preparation of Plant Material

Fruit were manually peeled and the rind portion was divided into small pieces. Rind

portion without network of membranes, was dried in shade with appropriate dust and dirt

control measures. Dried peel were subsequently ground to 40 mesh size for production of

peel powder. Finely ground pomegranate peel powder was packed in an airtight plastic jar

and kept in refrigerated conditions for further analysis (Negi and Jayaprakasha 2003).

3.3 Chemical Analysis of Peel

Pomegranate peel was subjected to following analytical procedures to determine

nutritional composition of the fruit waste.

1. Proximate Composition

2. Mineral Profiling

3. Total Phenolic Contents

3.3.1 Proximate Composition

3.3.1.1Moisture

Finely ground samples of pomegranate peel powders were subjected to drying in triplicates to

determine moisture contents of the dried plant material. A quantity measuring 5 g of sample was

taken in pre-weighed china dish and was dried in hot air oven (Memmert, Germeny ) at 105 oC ± 5

oC untill a constant weight (AOAC 2000). Peel moisture contents were interpreted through following

formula:-

45

Wt. of original sample – Wt. of dried sample

Moisture (%) = × 100

Wt. of original sample

3.3.1.2Crude protein contents

Micro kjeldahl distillation process (AOAC 2000) was followed to determine nitrogen

contents of the pomegranate peel powder. Peel powder samples were digested in a round

bottom long neck flask with H2SO4 in presence of digestion mixture for 3-4 hours till the

contents of digestion flask turned to transparent color. Samples were then diluted with

distilled water up to 250 mL in a volumetric flask. The ammonia generated from the samples

and trapped in H2SO4 was liberated by adding NaOH solution through distillation and

collected in a flask containing 4% boric acid solution. Methyl red was used as an indicator in

boric acid solution and nitrogen contents were measured by titration with 0.1N H2SO4

solution. The crude protein value was determined by using thefollowing formula.

0.0014 × Vol. of 0.1N H2SO4 × 250 ml

N % ═ × 100

Vol. of diluted sample × Wt. of original sample

3.3.1.3 Crude fat contents

Oven dried samples of pomegranate peel were subjected to crude fat extraction in

soxtec apparatus (Behr, Germany) using the method followed by AOAC (2000). An amount

measuring 2g of dried sample was packed in filter paper and was subjected to extraction

process by utilizing hexane as solvent. Extraction process continued for a period of 3-4 hours

and the flask contents were transferred to a pre-weighed dried petridish after considerable

recovery of the solvent and were subjected to high temperature in hot air oven to remove

residual solvent from the samples. Petridish was cooled in a desiccator and weighed for

calculating crude fat contents by using the following formula.

46

Weight of fat

Crude fat (%) = × 100

Weight of sample

3.3.1.4 Crude Fiber Contents

Oven dried and defatted samples of pomegranate peel were subjected to crude fiber analysis in

accordance with the standard crude fiber estimation process described by AOAC (2000). Sample

weighing 2 g was taken in 1000mL beaker, followed by an addition of 1.25% H2SO4 (200mL) and

was digested by boiling for a period of 3min. Digested material was filtered through whattman no. 42,

using vacuum pump assisted filtration assembly. The residues were washed with hot water at boiling

temperature to make the sample acid free. Alkali digestion was performed with in 200mL boiling

NaOH(1.25 %) for a period of 30min. Residues were again filtered, washed with hot water and

transferred to pre-weighed crucibles for drying and charring. Charred samples were ignited at a

temperature of 550-600oC for 5-6 hours to obtain ash. Crucibles were cooled and re-weighed and loss

in weight was recorded as crude fiber content of the sample. The crude fiber was calculated as under:-

Weight of residue – Weight of ash

Crude fiber(%) ═ × 100

Weight of sample

3.3.1.5 Ash Contents

An oven dried sample weighing 5g was transferred to a pre-weighed crucible and

charring was performed to obtain carbon free ash. Charred samples were subsequently

ignited in muffle furnace (Volcan) at 550 oC untill constant weight of ash (grey) was

obtained (AOAC 2000). Ash percentage in the samples was measured by using the following

formula.

Weight of ash

Ash contents (%) ═ × 100

Weight of sample

47

3.3.1.4 Nitrogen free extract (NFE)

NFE concentration in pomegranate peel was calculated by subtracting water,

carbohydrates, protein, fiber, fat and ash contents from 100 as has been given below.

NFE % ═ 100 – (moisture % + crude protein% + crude fiber %+ crude fat % + Ash

%)

3.3.2 Estimation of Minerals

Macro and microelements from dried pomegranate peel were estimated by using

Atomic Absorption Spectrophotometer (Thermo Scientific iCE 3000 series) and Flame

photometer (Model 410) Sherwood Scientific Ltd-UK) by using method described in

AOAC (2000).

A sample weighing 0.5g of pomegranate peel was wet digested in 100mL conical

flask by using wet digestion method (AOAC 2000). Initial digestion was performed with

10mL HNO3 at 70ºC for a period of 20min. Digestion was further proceeded at 190ºC by

using HClO4until the solution turned clear. Digested samples were diluted to 100mL with

deionized water and filtered to remove any un-dissolved or suspended solids.

Filtered digested samples were loaded on atomic absorption spectrophotometer and

flame photometer to determine mineral contents of the sample. Standard curve for each

element was prepared with standards of known strength and concentration of elements in

samples was determined accordingly.

3.3.3 Peel antioxidants profiling

3.3.3.1Extraction of peel phenolics

Phenolics from finely ground peel samples were extracted with different solvents including

ethanol, methanol, acetone and water. 70:30 solvent: water ratio was maintained for all

solvents except water extracts. Extraction was performed at 40ºC with homogenous

continuous shaking for a period of 3hrs. Extracts were filtered through whatman no. 41 and

the peel residues were again washed with the respective solvents to remove traces of

phenolics. Filterates were concentrated at Rotary evaporator (Heidolph, Hei-Vap, Germany)

at 25ºC. Concentrates of all extracts were freeze dried and the dried extracts were stored at -

70ºC in upright ultralow freezer (Sanyo, MDF-U32V, Japan) for further use.

3.3.3.2Total phenolic contents (TPC)

48

Total phenolic contents of peel extracts were determined by the method as adopted by Li et

al. (2006) using gallic acid as standard compound. Peel extracts were dissolved in methanol –

water solution and 0.5mL aliquots were pipetted in test tube. A volume measuring 2.5mL of

10 fold diluted Folin Ciocalteu Reagent (FCR) was added to the sample tubes followed by

addition of 2mL of 7.5% Na2CO3. Sample and the reaction mixture were allowed to stand for

30 min at 25ºC and the absorbance was subsequently read spectrophotometrically (UV-Vis

3000, ORI, Germany) at 760nm. Series of gallic acid standard with concentration ranging

from 10 – 100mg/L were run as standard concentration to plot the standard curve. Results

obtained were expressed as mg gallic acid equivalent (GAE) per 100g.

3.3.3.3Proanthocyanidins contents

Concentration of pomegranate peel proanthocyanidins was determined by the method

followed by Sun et al. (1998). 50mg/l sample solution was prepared with methanol – water

mixture. 0.5mL of the aliquot was drawn in a test tube and mixed with 3mL of vanillin (4%

prepared with methanol as solvent) and 1.5mL of HCl. Reaction mixture was allowed to

stand for a period of 15min at room temperature and the absorbance was measured at 500nm

using spectrophotometer (UV-Vis 3000, ORI, Germany). Catechin was used as standard and

the results were expressed as catechin equivalent.

3.3.4 Antioxidant Assay

3.3.4.1 Radical scavenging properties of pomegranate peel (DPPH Method)

Radical scavenging property of pomegranate peel extracts was determined by DPPH method

as described by Singh et al. (2002). Multiple concentration of peel extracts ranging from 50 –

100 µl (50 to 100mg/L) were prepared and pipetted in labeled test tubes. Volume of the tubes

was adjusted to 100µl by using methanol. 5mL of 0.1mM DPPH reagent, prepared with

methanol, was added to the contents of the test tubes and vortexed. Stay time of 20min was

given to the test tubes at 27ºC. Extract were replaced with methanol in control. Change in

absorbance was observed at 517nm. Free radical scavenging activity of methanolic,

ethanolic, acetone and water extracts was calculated by using following formula:-

% Radical Scavenging Activity = (Control OD - Sample OD/Control OD) × 100

49

3.3.4.2Ferric reducing antioxidant power (FRAP)

Ferric reducing antioxidant power of pomegranate peel extracts was determined by

the method adopted by Zahin et al. (2010) with some modifications. FRAP reagent was

prepared from TPTZ (10mM), ferric chloride (20mM) and sodium acetate buffer (pH 3.6) in

a ratio of 1:1:10. Reaction mixture was heated for 10min at a temperature of 37◦C and 300µl

of it was pipetted in a test tube. Powdered extracts weighing 25mg were taken in FRAP

reagent tubes and vortexed. Absorbance of samples and control was measured at 593nm

spectrophotometrically using (UV-Vis 3000, ORI, Germany). Ferrous sulphate was used as

standard and its multiple concentrations ranging from 100 – 1000µM were prepared.

Absorbance of the standard solution was accordingly read at 593nm. Results of the samples

and control were expressed as µM of Fe (ferrous ions to ferric ion conversion).

3.3.5 Antimicrobial assay of pomegranate peel extracts

Microorganisms including B. subtilis (ATCC 6633), E. coli (ATCC 25922), S. aureus

(ATCC 25923), P. aeruginosa (ATCC 27853), S. typhimurium (ATCC 14028) and A. niger

(ATCC 10575) were tested for antimicrobial activity. Antimicrobial assay of various PoP

extracts was performed by the method described by Al-Zorkey (2009). Freshly grown

cultures of test organisms were serially diluted and 1mL of diluted cultures carrying 106cfu

were added to sterile growth medium, i.e. 100 mL tryptose soya agar. Seeded growth

medium was poured to sterile petri plates after tempering to 45ºC. Wells of 6 mm diameter

were punched in solidified plates with a sterile borer. Pomegranate peel extracts (100µl) were

placed in wells and the plates were left for 30min at room temperature for diffusion of

extracts into the growth medium. Potato dextrose agar was used as growth medium for

antifungal activity. Fungal suspension measuring 100µl was spread onto the surface of

solidified potato dextrose agar plates and wells were prepared for dispensing liquid extracts

as described earlier. Ampicillin and fluconazole were used as positive control and plates were

incubated at 37ºC and 27ºC for a period of 24 – 48h for bacterial and fungal growth,

respectively. Negative controls were set for solvents (ethanol, methanol and acetone) to

determine their possible microbial inhibitory effects. Visible growth of test organism was

identified at control plates and zone of inhibition (mm) were measured for pomegranate peel

extracts derived via water, ethanol, methanol and acetone extracts.

50

Twenty microliter of crude PoPx with various dilutions ranging from 0.1 –

100mg/mL were pipetted in 96-well microtitre plates. 160µl of nutrient broth (for bacteria)

and sabouraud’s dextrose broth (for fungi) were added to the wells along with 20µl of

microbial cultures (1 × 104cfu/mL) of test strains. Microplates with bacterial cultures were

incubated at 37ºC for 24hrs while those with fungal cultures at 27ºC for 72hrs. Ampicillin and

fluconazole were used as standard drugs and minimum inhibitory concentration (MIC) was

calculated as the lowest concentration of extracts showing microbial growth inhibition (Atta-

ur-Rehman et al. 2001).

3.3.6 Urease Inhibition Activity

A reaction mixture comprising jack bean urease enzyme (25μl) and urea buffer (55µl)

were incubated in 96-well plates with 5μl peel extracts at 30ºC. Ammonia production was

determined to assess urease activity by adding 45μl of phenol reagent and 70μl of alkali

reagent in each well. Absorbance was measured on a microplate reader after 50 min. pH of

the test sample was maintained at 6.8 and thiourea was used as standard urease inhibitor.

Urease inhibition activity of the tested samples was determined as hereunder:-

Urease inhibition activity (%) = 100 – (ODs /ODt) × 100

Where ODs was absorbance of sample and ODt was for thiourea absorbance.

3.4 Product Development

Pomegranate peel, peel extracts and peel meal were supplemented in widely available

and economical food product i.e. cookies. Cookies were prepared by adopting some

modifications in the method no. 10-50D described in AACC (2000). Standard recipe of

cookies followed in the experiment has been described in Table 3.1. Modifications were

adopted in flour quantity as replacement of straight grade flour with pomegranate peel

extracts, peel and peel meal powder were made in different proportions as shown in Table

3.2.

51

Table 3.1: Standard recipe of cookies

Flour/Supplemented flour 500g

Sugar 250g

Hydrogenated vegetable ghee 250g

Eggs 3 no.(Avg. wt. 60g each)

Baking powder 10g

Table 3.2: Supplementation levels of PoP, PoPx and PM in cookies

Product Cookies supplemented with

PoP PM PoPx

T0 0 0 0

T1 1.5 1.5 0.25

T2 3 3 0.50

T3 4.5 4.5 0.75

T4 6 6 1.0

T5 7.5 7.5 2.0

PoP = Pomegranate peel powder

PM = Pomegranate peel meal

PoPx = Pomegranate peel extracts

3.5 Cookies

3.5.1 Preparation of cookies

The ingredients were weighed accurately and vegetable ghee (hydrogenated fat) and

sugar were mixed and eggs were added one by one. The straight grade flour along with pre-

weighed proportions of PoP and PM were homogenously mixed and blended with baking

powder and sifted. Peel extracts were weighed and dissolved at 30ºC in melted hydrogenated

fat. The flour and peel powder composite were added to sugar-ghee-egg mass and mixed to

get a homogeneous mass. The batter was rolled out to uniform thickness with the help of the

rolling pin. Cookies were cut out with the help of cookie cutter and placed in trays. Baking

52

was done at 170–180ºC for 15–20min. Cookies were allowed to cool at room temperature for

8–10min and stored in airtight glass jars for further analysis.

3.5.2 Sensory evaluation

Pomegranate peel, meal and extracts supplemented cookies were evaluated for

sensory characteristics i.e. taste, color, crispiness, texture and overall acceptability on 9-point

Hedonic Scale (Land and Shepherd 1988). Fifty sensory panelist were selected based on their

product discriminative ability for different sensory attributes. The objectives of the study

were briefed to the panelists and the judges were given questionnaires to record their

observations. The information contained on the proforma was; 9 = like extremely; 8 = like

very much; 7 = like moderately; 6 = like slightly; 5 = neither like nor dislike; 4 = dislike

slightly; 3 = dislike moderately; 2 = dislike very much; 1 = dislike extremely. Sensory testing

was made in the panel room completely free of food/chemical odor, unnecessary sound and

mixing of daylight (Akhtar et al. 2008).

3.6 Product stability study

3.6.1 Microbiological quality of cookies

Evaluation of supplemented cookies for total bacterial counts and yeast/mould growth

was made fortnightly in line with the method described by APHA (1992). Cookies, being

stored in sterile air tight containers were used in microbiological evaluation. Samples were

ground with sterile pestle mortar in laminar air flow. A quantity weighing 1g of powdered

sample was taken in sterile test tube and dissolved in 9mL ringer solution. Test tubes were

caped and centrifuged for a period of 5min to decant fat layer from the sample. 10fold

dilution was made for further evaluation of total bacterial and yeast/ moulds load of the

samples. Nutrient agar and potato dextrose agar were prepared in accordance with the

instructions provided by the manufacturer. Pour plate method was adopted in determining

total plate counts however; solidified sterile plates of potato dextrose agar were used in

yeasts and moulds counts estimation by spread plate method. Sample inoculated nutrient agar

plates were stored at 37ºC for 24 – 48h while those of potato dextrose agar were incubated at

25◦Cfor a period of 5 days. Microbial counts were recorded after 48h (total plate counts) and

5 days (Yeast/Moulds) by using colony counter.

53

3.6.2 Free fatty acid levels of cookies

Cookies stored for a period of four months in air tight glass jars were periodically analyzed

for free fatty acids contents at an interval of 02 months. n-hexane was used to remove fat

from the crushed cookies and the extracted lipids were analyzed for free fatty acid contents

according to the method described in AOCS (1993).

3.6.3 Total phenolics and antioxidant activity of cookies extracts

PoP, PM and PoPx supplemented cookies samples were converted into fine powder

by using pestle and mortar. About 5g of powdered sample was extracted with 100mL of

deionized water for a period of 1h. Extraction was performed at 25ºC in orbital shaker at

stirring speed of 250rpm. The liquid extracts were filtered through Whatman no. 41 and re-

extraction of the residues was performed with deionized water. Filtrates were pooled and

concentrated by rotary evaporator (Heidolph, Schwabach, Germany) at 25ºC. Total phenolic

contents of cookies extracts were determined by Folin-Ciocalteu method as adopted by Li et

al. (2006) with some modifications using gallic acid as standard. Absorbance was measured

spectrophotometrically at 725nm and results were expressed as mg gallic acid equivalent

(GAE). 2,2-diphenyl-1-picrylhydrazyl (DPPH) equivalent antioxidant activity of

supplemented cookies extracts was determined by the method as adopted by Singh et al.

(2002). Absorbance was measured spectrophotometrically at 517nm and % DPPH

scavenging activity was calculated by using following formula:

% Radical scavenging activity =(Blank OD - Sample OD/Blank OD)× 100

Ferric reducing antioxidant power (FRAP) of supplemented cookies extracts was

measured by the method of Benzie & Strain (1996). Chromogenic ferrous compound formed

by reduction of ferric tripyridaltriazine complex was measured at 593nm. Calibration curves

were constructed from various concentrations (100–1000mmol/L) of FeSO4.7H2O. Results

were expressed as concentration of antioxidants bearing ability to reduce ferric equivalent to

1mmol FeSO4.

3.6.4 Lipid Peroxidation by Thiobarbituric acid Assay

Thiobarbituric reactive substances assay was performed to determine lipid

peroxidation of cookies. Cookies were ground to powder and homogenized sample (0.5g)

was extracted with 25mL of water – ethanol mixture (20:80 v/v). Extracts recovered from

54

PoP and PoPx supplemented cookies, measuring 2mL,were drawn in test tubes already

containing 2mL of trichloroacetic acid (20%) and butylated hydroxyl toluene (0.01%). Test

tubes were subjected to water bath at 70◦C for a period of 30 min. Contents of the test tubes

were cooled and centrifuged for 10min at 1300g.

3.7 Toxicological evaluation of pomegranate peel, meal and peel extracts

3.7.1 Cytotoxicity/ brine shrimps lethality test

The test was carried out using the method as outlined by Carballo et al. (2002).

Freeze dried PoP extracts (20mg) extracted with methanol, ethanol, acetone and water were

dissolved in 2mL of the respective solvent. PoPx, measuring 5, 50 and 500µl equivalent to

10, 100 and 1000µg/mL were placed in individual vials and the solvent in each sample vial

was allowed to dry overnight. 10 nauplii (shrimps larvae) were transferred to each vial with a

pasteur pipette and volume of the vial was made to 5mL with sea water. Larvae were

incubated for 24h with illumination at 25 – 27 ◦C. Negative and positive control (solvent and

cytotoxic reference drug i.e. Etoposide) was also run parallel to the samples. Numbers of

dead larvae were counted after an incubation period of 24h. Mortality was calculated by

using the following formula:-

Mortality % = [1- (A1-A2)/A1] × 100

whereA1= Live control (without sample)

A2 = Death in presence of the sample

3.8 Efficacy study

Efficacy study was conducted to evaluate any suspected toxicity of pomegranate peel,

peel meal and peel extracts already tested in organoleptically accepted cookies. Corn starch

was replaced with PoP, PM and PoPx to incorporate designed doses of PoP, PM and PoPx in

animal diet.

55

3.8.1 Treatments

Treatment combinations selected for efficacy studies are given hereunder:-

Table 3.3: Treatment combinations of PoP, PM, and PoPx selected for efficacy studies

Treatment

Animal Groups

PoP

(g/100g)

PM

(g/100g)

PoPx

(g/100g)

T0 0 0 0

T1 2.0 2.0 0.25

T2 4.0 4.0 0.50

T3 6.0 6.0 1.00

3.8.2 Procurement of rats

One hundred male albino rats with an average weight of 144g were procured from

Animal Division, University of Lahore-Pakistan. Rearing of animal was carried out at the

animal room of the Institute of Food Science and Nutrition with the approval from Ethical

Review Committee of the University.

3.8.3 Experimental design

Animals were divided into three groups (PoP, PM, PoPx each with three treatments)

and a control (Table 3.3). Nine healthy male rats were grouped for each treatment while the

control independently was comprised of 09 healthy rats. Animals were acclimatized to the

environment and fed for a period of one week on basal diet. Three animals from each

treatment group were periodically sacrificed at 0, 28 and 56 days interval to assess effect of

PoP, PM and PoPx on serum chemistry and other hematological parameters.

3.8.4 Preparation of diet

Animal diet was prepared according to Lavrat – Vernny (1999) model. Following standard

recipe was used for preparing basal diet of the animals-

56

Table 3.4: Standard recipe for basal diet of animal

Ingredient g/100g

Corn starch 65.5

Casein 10

Cellulose 10.5

Corn oil 10

Mineral Mixture 3

Vitamin Mixture 1

Pomegranate peel, peel meal powder and powdered extracts were added to the recipe by

equal replacement of corn starch from the animal standard diet formulation in following

proportions:-

Table 3.5: Supplementation of PoP, PM, and PoPx by replacement of corn starch

Ingredient g/100g PoP PM PoPx

T0 T1 T2 T3 T1 T2 T3 T1 T2 T3

Corn starch 65.5 63.5 61.5 59.5 63.5 61.5 59.5 63.25 63.0 62.5

Casein 10 10 10 10 10 10 10 10 10 10

Cellulose 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5

Corn oil 10 10 10 10 10 10 10 10 10 10

Mineral Mix 3 3 3 3 3 3 3 3 3 3

Vitamin Mix 1 1 1 1 1 1 1 1 1 1

PoP 0 2.0 4.0 6.0 0 0 0 0 0 0

PM 0 0 0 0 2.0 4.0 6.0 0 0 0

PoPx 0 0 0 0 0 0 0 0.25 0.50 1.0

One Sq. ft. wire bottom cages with ample ventilation were used to adequately house 4

animals in each cage. Animal room temperature was maintained at 25 - 30ºC with 12h light

and dark period. Proper labeling was performed to identify different treatments in each

57

group. Rats were provided with basal diet for a period of one week followed by experimental

diet for 28 days and another 28 days. Clean and safe drinking water was provided ad libitum

to the experimental rats.

3.8.5 Physical parameters

Feed and water intake was determined for the animals of each cage daily in entire

study period however; body weight of each rat was measured on weekly basis. Random

Blood Glucose level of each rat was also determined at one-week interval.

3.8.6 Sampling procedure

At 0, 28 and 56 day of feeding, one third of the sample population was randomly

isolated from their respective groups and treatment. Animals were restricted from the diet for

a period of 8 h before sampling and anesthesia was performed with pentobarbital. Final

weight and blood glucose level of each animal was recorded before anesthesia. Rats were

humanly sacrificed one by one and aortic blood was taken in labeled vacutainer containing

3.8% EDTA solution. A part of the blood was preserved for hematological study while the

remaining was centrifuged at 7500rpm for 2min to collect plasma. Plasma samples were

collected in eppendorf tubes, labeled and refrigerated in upright freezer (MDF-U33V-PB,

Japan) at -70ºC for further analysis. Animals were dissected to remove organs and tissues

including heart, lungs (right and left), liver, kidneys (right and left) and spleen. Each organ

was weighed individually and preserved at -70ºC in polyethylene bags. Visual examination of

individual animal was also performed to identify any symptomatic sign of toxicity on animal

general health, behavior and individual organ.

3.8.7 Plasma analysis

3.8.7.1 Plasma Biochemistry

Rats’ plasma was tested for glucose (mg/dL), triglycerides (mg/dL), cholesterol

(mg/dL), low density lipoprotein (LDL) (mg/dL) and high density lipoprotein (HDL)

(mg/dL), serum total protein, albumin, serum creatinine, serum bilirubin, serum uric acid,

Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate transaminase (AST)

were estimated with commercial kits (Merck, Germany) according to the following working

procedures as adopted by Ogunlana (2012).

58

3.8.7.2 Estimation of plasma glucose levels

Plasma glucose levels in blood samples were estimated by colorimetric method

working on Trinder Reaction principle. A volume measuring 1000µl working standard was

drawn in a blank test tube along with the one labeled as standard and sample tubes. A volume

of 10µl of standard and 10µl of sample i.e. plasma was taken in previously prepared standard

and sample tubes respectively.

Contents of each tube were mixed homogenously and stored at 37ºC for 90seconds.

Absorbance was recorded at 505 nm and the concentration of plasma glucose was determined

as given below:-

Sample absorbance

Plasma glucose (mg/dL) = × Standard concentration

Standard absorbance

3.8.7.3 Determination of plasma cholesterol

Commercial diagnostic kits (Merck) were used to calorimetrically determine plasma

cholesterol levels in rats. Working solutions were prepared by gently mixing one vial of

reagent with buffer solution. Standard solution along with sample and 20µl distilled water

were pipetted in a test tube and 2mL of working reagent was added to the tube. Test tubes

were homogenously shaken and the resultant homogenates were incubated at 37ºC for 5min.

Absorbance was recorded for the both known standard and samples at 505nm. Standard

solution was prepared of 200mg/dL strength. Plasma cholesterol was determined in

accordance with the following formula:-

Sample absorbance

Cholesterol (mg/dL) = × Standard concentration

Standard absorbance

59

3.8.7.4 Determination of HDL-cholesterol

Commercial diagnostic kit (Merck) was used for the determination of HDL-

cholesterol. A quantity measuring 200 µl plasma was taken in a test tube along with 500 µl of

diluted reagent. Ingredients were homogenously mixed and were allowed to stand at room

temperature for a period of 10min. Suspended samples with reagent were centrifuge at

4000rpm for 10min. 70 µl supernatant was collected in another test tube from the centrifuged

from above mentioned working solution. 2mL of cholesterol reagent was added as working

reagent in the supernatant containing test tubes. Blank and known standard were also

prepared to get quantitative value for HDL- cholesterol. Absorbance of known standards

strength and unknown samples was read at 505nm. HDL cholesterol was calculated as given

below:-

Sample absorbance

HDL – Cholesterol (mg/dL) = × Standard concentration

Standard absorbance

Standard concentration was set as 200mg/dL

3.8.7.5 LDL-Cholesterol (mg/dL) calculation

LDL – cholesterol was calculated by using following formula:-

LDL – Cholesterol (mg/dL) = Total Cholesterol – HDL Cholesterol – Triacylglycerols

5

3.8.7.6 Determination of triglycerides (mg/dL)

Serum triglycerides levels were determined by using commercial diagnostic kits

(Merck). 10µl of standard solution was pipetted in 3 test tubes labeled as standard, sample

and blank and 10µl of sample was added in sample test tube. Working reagent (1000µl) was

added in each test tube and tube contents were mixed homogenously. Test tubes were

incubated at 37ºC for 5min and absorbance was read at 500nm against known standard

solution. Calculations were carried out as under:-

60

Triglycerides (mg/dL) = (Abs of sample / Abs of standard) × Conc. of standard

3.8.7.7 Determination of total plasma protein (mg/dL)

Plasma total protein (mg/dL) was measured colorimetrically by using diagnostic kits

(Merck). Biuret reagent measuring 2.5mL)each was taken in labeled test tubes (sample, blank

and standard) and 0.05ml of sample, distilled water and standard solution were added to these

test tubes, respectively. Tubes were incubated at 25–30◦C for 25min and absorbance was

recorded against standard and blank at 540nm. Calculations were carried out as hereunder:-

Total protein (mg/dL) = (Abs of sample / Abs of standard) × Conc. of standard

3.8.7.8 Determination of albumin (g/dL)

Colorimetric technique was adopted to determine plasma albumin concentration. A volume

measuring 3ml of BCG reagent was pipetted in each blank, standard and sample test tube.

0.01mL of distilled water was added to blank while the same quantity of standard and sample

was poured in standard and sample tubes, respectively. All components of test tubes were

mixed homogenously and incubated at 20–30 ◦C for 25min. Absorbance of standard and

sample was read against blank at 630nm. Plasma albumin was calculated as hereunder:-

Albumin (g/dL) = (Sample Abs. / Standard Abs.) × Standard Conc.

3.8.7.9 Serum bilirubin

Commercial kits were used for determination of serum bilirubin concentration in rat

models. 200µl of reagent 1 carrying sulphanilic acid and HCl, 200µl of sample and 1000µl of

R3 carrying caffeine were added to each test tube allocated as sample, standard and blank.

Sample tube was pipetted with 50µl of reagent 2 i.e. sodium nitrite while same amount of

distilled water was pipetted in blank tube. Test tubes were vortexed and incubated at room

temperature for a period of 10min and 1000µl of reagent 4 i.e. Tartrate and NaOH was added

in each tube. The aliquots were again mixed and incubated for further 20min at room

temperature (25◦C). Absorbance of sample and standard were measured at 578nm and

bilirubin contents were calculated by using following formula.

Bilirubin concentration = Sample absorbance ×10.8

61

3.8.7.10 Serum enzymes levels

Serum enzymes including serum glutamate pyruvate transaminase (SGPT) and serum

glutamate oxaloacetate transaminase (SGOT) were determined at both 28 and 56 days

intervals.

3.8.7.11Determination of serum glutamate pyruvate transaminase (SGPT)/ALT

Standard Curve: Colorimetric method was used to determine SGPT by using commercial

diagnostic kits (Merck).Six test tubes labeled as 1, 2, 3, 4, 5 and 6 carrying 1000µl distilled

water each, was added with various concentrations i.e. 500, 450, 400, 350, 300, 250 of SGPT

substrate. Standard reagent measuring 50, 100, 150, 200 and 250µl were added up to

substrates tubes. Five hundred microlitre color reagent was added in each tube and were

allowed to stand for 20min after a uniform vortex. Five ml of 0.4N NaOH was finally added

to the test tubes and absorbance (at 505nm) was recorded after a stay period of 15min.

Sample Analysis: A test tube carrying 500µl SGPT substrate was incubated for 5min at 37ºC

and 100µlsample was pipetted in it along with 500 µl of color reagent and incubated for

20min at room temperature. A further 15min incubation time at room temperature was

assigned the sample tube after adding 5ml 0.4N NaOH. Absorbance was measured at 505nm

against a water blank. SGPT Values for the samples were derived by incorporating sample

absorbance values in standard curve.

3.8.7.12 Determination of serum glutamate oxaloacetate transaminase (SGOT)/AST

Colorimetric method was used to determine SGOT by using commercial diagnostic kits.

Standard Curve: Six test tubes labeled as 1, 2, 3, 4, 5 and 6 carrying 1000µl distilled water

each was added with various concentrations i.e. 500, 450, 400, 350, 300, 250 of SGOT

substrate. Rest method adopted was same as mentioned for SGPT standard curve.

62

Sample Analysis: SGPT substrate was replaced with SGOT while the entire procedure was

same as adopted for SGPT sample analysis and SGOT activity was calculated by

incorporating absorbance reading in standard curve.

3.8.7.13Determination of serum creatinine

Standard commercial kits (Merck) were used for estimation of creatinine in rats

serum. Three test tubes, individually carrying 100 µl plasma, 100 µl creatinine standard and

100 µl distilled water and 1000 µl working reagent (carrying equal volume of picric acid and

NaOH). Test tube contents were vortexed and 30second stay time was given to the contents.

Absorbance of sample (A1sample) and standard (A1standard) were measured at 492nm against

reagent blank. Standard and sample readings were again taken after a stay time of 2min and

absorbance was represented as (A2 sample and A2standard) and creatinine levels were calculated

by using standard formula.

A2sample – A1sample

Creatinine = × standard creatinine conc.

A2standard – A1standard

3.8.7.14Determination of serum uric acid

Standard commercial kits (Merck) were used for estimation of uric acid in rats serum. Three

test tubes vis. standard, sample and blank were individually pipetted with25µl standard, 25µl

plasma and 1.0mL mono-reagent solution, respectively. The aliquots of test tubes were

allowed for 10min incubation at room temperature and absorbance of both the sample (A

sample) and standard (A standard) were measured at 520nm. Uric acid levels in plasma were

calculated by using following formula:-

Asample

Uric acid = × standard uric acid conc.

Astandard

63

3.8.8 Hematological Analysis

Hematological analysis of rats blood samples was performed to determine red blood

cells and white blood cells indices including red blood cells counts, hemoglobin, hematocrits

concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, white

blood cells, platelets, lymphocytes and neutrophils by using automatic hematology analyzer

following the procedure as described by Dacie and Lewis (1984).

3.9 Statistical Analysis

Each analysis was performed in triplicate and results of analyses were expressed as

mean ± S.E and biological activities were evaluated by analysis of variance technique

(ANOVA) using program Statistix 8.1. Test of normality was performed by Shapiro-Wilk

test and two way ANOVA was used for analyzing animal modeling / safety study data.

Pearson correlation was performed among correlation variables. Difference among means of

various treatments was analyzed by Latin Square Design (LSD) at 95% p<0.05 confidence

interval (Steel et al. 1997).

64

Chapter 4 Results & Discussions 4.0 Preamble

This section deals with the biological properties of PoP and PoPx derived from white

rind Alipuri cultivar of pomegranate. The study comprehensively describes nutritional

properties of PoP and its potential for being utilized as a functional ingredient in cookies.

Results obtained through the present study establish PoP potential as a novel ingredient in

product development and its role as a functional component to impart lipid stability and

microbial inhibitory properties in food preparations. This section further explains various

facets of safety assessment of PoP, PoPx and PM to determine any of the suspected toxicity

of the pomegranate peel, its extracts and the meal at various supplementation levels.

4.1 Extracts yields and total phenolic contents of pomegranate peel fraction

The levels and components of phenolics recovered from natural products depend

upon the particle size of the plant material, the type of solvent, the solvent – solid ratio, and

extraction temperature (Akhtar et al. 2015). Hydro-alcoholic extraction yields of PoPx from

various solvents and total phenolic content of extracts are presented in Table 4.1. Highest

extract yield was obtained from ethanol (24.23%) followed by acetone (21.2%), methanol

(20.81%) and water (14.36%). Hydro-alcoholic extraction at a ratio of 30:70 was found to be

a viable strategy to improve extracts recovery. Total phenolic contents of PoP extracts ranged

between 273.5 – 427.19mgGAE/g on a dry weight basis indicating an elevated phenolics

profile of the inedible fruit fraction. Irrespective of extract yield, use of acetone resulted in

the highest yield of total phenolics from PoP.

The higher phenolic levels profile of acetone, methanol and ethanol extracts indicates

the presence of medium polar to polar compounds, predominantly phenolic acids, flavonoids,

sugars and polysaccharides in plant material (PoP). One group of researchers recently

demonstrated solvent concentration and extraction time to be the contributory factors in

increasing the total phenolics concentration from pomegranate husks, i.e. ~324.9mgGAE/g

(Amyrgialaki et al. 2014).

65

Similarly, the present study confirmed greater extraction and yield of phenolics from

PoP with a 0.2 mesh size using hydro-alcoholic phenolic extraction at 25ºC. Comparative

analysis of extracts and yield of phenolics in this study further suggest that extraction of PoP

is superior if carried out using a particle size of 0.2mm at 25ºC as compared to those

procedures with a particle size of ~3.5mm at temperature ranging from 40 – 95ºC (Qu et al.

2010).

Dried extracts of Alipuri cultivar were shown to contain increased phenolic contents

with relatively higher yields of total phenolics, i.e. 427.19mg GAE/g as compared to earlier

studies from Iran where the levels of total phenolics were estimated to be 220.1mgGAE/g

from sweet white cultivars (Ardekani et al. 2011). Trends in phenolics yield were

comparable to the findings of Negi et al. (2003) indicating acetone and water as the highest

(520mg/g) and the lowest (48mg/g) recovery media for phenolics, respectively.

Proanthocyanidins

Proanthocyanidins are oligo and polymeric flavan – 3 – ol condensed tannins. Despite

their ability to chelate mineral ions, proanthocyanidins are widely known to act as reducing

agents to prevent oxidative stress and associated disorders (Scalbert et al. 2000). With

respect to their potential in ameliorating oxidative stress, levels of proanthocyanidins were

monitored for their concentration in PoP hydro-alcoholic extracts. Significantly higher yield

of proanthocyanidins was recovered from PoPx of Alipuri cultivar (Table 4.1).

As with their capacity to recover maximum phenolics contents, PoP acetone / water

extracts also exhibited the highest levels of proanthocyanidins i.e. 17.47 mgGAE/g.

Comparatively lower levels of proanthocyanidins were recovered by pure water

(16.13mgGAE/g) followed by ethanol / water extracts (12.13mgGAE/g) indicating increased

affinity of proanthocyanidins towards solvents polarity. The enhanced ability of

acetone/water to extract proanthocyanidins has also been reported by Zam et al. (2012)

indicating a better tendency of polar solvents to extract oligomeric and polymeric

proanthocyanidins.

PoP extracted with a combination of methanol, ethanol and acetone was previously reported

resulting in a comparatively reduced yield of proanthocyanidins (10.9mg/g; Li et al. 2006) as

66

compared to the results obtained in the present study. A linear correlation between

proanthocyanidins concentration and free radicals scavenging properties of PoPx were

reported by El kar et al. (2011) suggesting proanthocyanidins as principle contributors to the

antioxidant content of PoPx (Table 4.1)

67

Table 4.1: Extracts yield, total phenolic contents, proanthocyanidins and antioxidant properties of PoPx at 25ºC

PoPx Yield (%) Total phenolic contents

mgGAE/g

Proanthocyanidins contents

(mgGAE/g)

Aqueous 14.36±0.07d 273.50 ± 1.30d 16.13±0.03b

EtOH 24.23±0.06a 361.79 ± 1.37c 12.13±0.05c

MeOH 20.81±0.07c 367.91 ± 1.50b 9.47±0.06d

AcN 21.2±0.05b 427.19 ± 2.36a 17.47±0.06a

Values sharing same letters are non- significant at p<0.05

EtOH = 70:30 (ethanol, water), MeOH = 70:30 (methanol, water), AcN = 70:30 (acetone, water), RSA = Radical scavenging activity

Table 4.2: Antioxidant activity of pomegranate peel aqueous, ethanolic, methanolic and acetone extracts

PoPx

Antioxidant activity

FRAP

(mmol/g)

DPPH

(%RSA)

Aqueous 61.27 ± 0.09d 84.47 ± 0.51c

EtOH 78.52± 0.15c 89.55± 0.68b

MeOH 80.31± 0.07b 90.49± 0.53ab

AcN 91.40± 0.16a 92.32± 0.79a


EtOH = 70:30 (ethanol, water), MeOH = 70:30 (methanol, water), AcN = 70:30 (acetone, water), RSA = Radical scavenging activity

68

4.2 Free radicals scavenging properties of PoPx

Oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity

(TEAC) and ferric reducing antioxidant power (FRAP) are considered reliable and widely

accepted assay techniques for the determination of total antioxidant activity (Lim and Lim,

2013). Based on their simplicity and analytical speed, free radicals scavenging properties of

PoPx against oxidants were measured by FRAP and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)

assays (Table 4.2). Hydro-alcoholic extracts showed greater free radical scavenging potential

as compared to PoP extracted by other media.

Comparing assay techniques, FRAP assay was found to be more effective for

acetone extracts, showing maximum free radical scavenging activity (91.40mmol/g) whereas

water extracts exhibited 32.5% lower total antioxidant activity than delivered by acetone

extracts (Table 4.2). Significant difference (P<0.05) in FRAP values was noticed among

acetone, methanol and ethanol extracts and the antioxidant activity of these extracts ranged

between 78.52 – 91.4mmol/g. Previous studies indicated that the DPPH assay was superior

in assessing inhibition by biological substrates of pre-formed radicals (Chen et al. 2013). The

current study validated these findings when applied to extracts of PoPx. Likewise, with the

FRAP assay, a positive correlation was also noticed between phenolic contents and DPPH

activity, i.e. an increase in inhibition was linked with increased levels of PoPx phenolics.

The DPPH scavenging activity of PoP acetone extracts (92.32%) was higher than

methanol (90.49%) followed by ethanol (89.55%) and water extracts (84.47%). The principle

role of PoP phenolics in inhibiting oxidative stress is generally attributed to their ability to

donate electrons to free radicals thereby converting them to relatively stable form (Akhtar et

al. 2015).

Hydro-alcoholic extracts of PoP were previously reported to contain substantial

concentration of total phenolics and have shown increased antioxidant activity i.e. ~ 438mg/g

and 93%, respectively (Malviya et al. 2014). The present study confirms that the indigenous

white pomegranate cultivar of Pakistan is a significant source of phenolics and hence could

potentially be exploited as potent free radical scavengers in biomedical formulations.

69

4.3 Urease inhibitory/antiulcer properties of PoPx

Proton pump inhibitors (PPIs) are pharmacological drugs for suppressing gastric acid

production and associated peptic ulcers (Forgacs and Loganayagam 2008). Because of their

antioxidant properties, plant polyphenolics have been reported to act as gastric protective /

remedial agents and hence are treated as alternate medicine for the treatment of gastric ulcers

(Sumbul et al. 2011).

Microbial ureases have innate potential to hydrolyze urea in its basic components i.e.

NH3 and CO2. Urease activity associated with some infectious microorganisms can

reportedly contribute towards onset of gastric ulcers, pyelonephritis and generation of urinary

stones (Mobley and Hausinger 1989).Despite its exceptional phenolic profile and antioxidant

potential, little information is available on the inhibitory properties of PoP and its extracts

against urease.

I investigated PoPx as a source of compounds inhibitory to urease (Table 4.3). Water

and acetone extracts of PoPx inhibited jack bean urease by 91.2 to 97.9% respectively. The

IC50 of PoPx extracted with acetone, ethanol, water and methanol against thiourea was

recorded as 30.0, 35.3, 41.6 and 44.4µM, respectively. A positive correlation (r2 = 0.716,

p<0.29) was noted among DPPH radical scavenging properties and urease inhibition.

Previousstudies (Nabati et al. 2012) on the inhibitory properties of PoP against urease

indicated higher IC50 values i.e. 1484µg/mL compared with the values reported in the

present study

The urease inhibitory concentration of PoPx observed here was ~47% higher than

those reported from 137 traditional medicinal plants (Nabati et al. 2012). These results

further demonstrate the ability of PoPx to inhibit urease production at a greater extent as

compared to a wide range of the popular medicinal plants, thereby validating the protective

role of PoPx antioxidants as proton pump inhibitors.

70

Table 4.3: Urease inhibition activity of pomegranate peel extracts

PoPx IC50 ± SEM (µM) Inhibition (%)

Aqueous 41.6 ± 1.46b 91.2

EtOH 35.3 ± 1.54c 96.2

MeOH 44.4 ± 1.36a 95.5

AcN 30 ± 0.05d 97.9


EtOH = 70:30 (ethanol, water), MeOH = 70:30 (methanol, water), AcN = 70:30 (acetone, water)

4.4 Antimicrobial properties of PoPx

Microbial diseases deleteriously affect a significant portion of the population from

developing countries and are thought to be underlying cause of morbidity and mortality.

Multi drug resistance in microbial strains continues to increase as antimicrobial agents are

becoming less effective (Akhtar et al. 2014). An innovative addition to the array of existing

antimicrobials would be broad – spectrum, natural biomolecules. Phytochemicals from

pomegranate have shown promising efficacy against multidrug resistant gram negative and

gram positive microorganisms (Gullon et al. 2016).

Our study confirmed that extracts of PoP of White Alipuri cultivar possesses exceptional

antimicrobial activity (Table 4.4) as all of the polar solvents demonstrated antimicrobial

activity against gram positive and gram negative, potentially pathogenic bacteria.

Hydroalcoholic extracts of PoP exhibited significant inhibition against Bacillus subtilis,

with zones of inhibition measuring 21.3mm from acetone extracts, 46% of those generated

ampicillin (Table 4.4 & 4.5). Similarly, acetone extracts of PoP inhibited Staphylococcus

aureus (19.7mm), Pseudomonas aeruginosa (17.5mm), Escherichia coli (19.3mm),

Salmonella typhimurium (15.7mm) and Aspergillus niger (19.3mm). However, decreased

microbial inhibition (P<0.05) was observed with PoP water extracts and smaller zones of

inhibition were noticed against all the tested microorganisms thereby indicating relatively

decreased concentration of antioxidants in these extracts. Relatively greater microbial

inhibitory potential was identified for both water / methanol and water / ethanol extracts that

suggested a correlation between higher concentration of PoP phenolics (hydrophilic and

hydrophobic) and antimicrobial activity (Table 4.4).

71

Health implications associated with Aspergillus niger toxicogenic nature are of

serious concerns in food safety. Larger zones of inhibition appeared on A. niger plates treated

with 70% acetone extracts of PoP and were found to be ~84% of those produced by

fluconazole. Convincingly, A. niger inhibitory potential of PoP water / acetone extracts

suggest its potential role as an ingredient of choice in formulating phytosanitory products.

Minimum inhibitory concentrations (MIC) of PoPx were determined for all tested organisms

(Table 4.5) and significantly lower doses (mg/mL) of peel extracts were found responsible

for inhibition of B. subtilis (0.25 – 0.43mg/mL) and S. aureus (0.36 – 0.66mg/mL). These

results are suggestive of greater tendency of PoP phenolics to permeate through microbial

cells of gram positive bacteria and interfere with microbial proteins’ sulfhydryl groups.

Relatively higher doses of PoP water extracts (0.43 – 1.30mg/mL) were required to inhibit

tested microorganisms implying relatively lower total phenolics profile of these extracts.

The antimicrobial potential of PoPx varies with respect to cultivar and genotypic

phytochemical diversity. A concentration of 0.2 – 0.78mg/mL of PoPx have been previously

reported as MIC against a wide array of gram positive and gram negative microorganism

(Fawole et al. 2012). Our results are in agreement with the information on antimicrobial

properties of PoP available in the literature, reporting a broad MIC range, i.e. 0.02 –

3.2mg/mL against microorganisms of human health significance (Duman et al. 2009; Choi et

al. 2011; Panichayupakaranant et al. 2010; Sadeghian et al. 2011).

Table 4.4: Antimicrobial activity of standard drugs

Microorganism Zone of inhibition of Ampicillin

(mm)

Zone of inhibition of Fluconazole

(mm)

B. subtilis 46 -

E. coli 27 -

S. aureus 42 -

P. aeruginosa 25 -

S. typhimurium 31 -

A. niger - 23

Values are the mean of replicates (n = 3)

72

Table 4.5: Antimicrobial activity of pomegranate peel extracts

PoPx Zone of inhibition (mm ± SEM)

B. subtilis E. coli S. aureus P. aeruginosa S. typhimurium A. niger

Aqueous 11.6 ± 0.05d 11.5 ± 0.15d 13.2 ± 0.05d 12.6 ± 0.05d 10.2 ± 0.05d 11.1 ± 0.05d

EtOH 17.5 ± 0.15c 16.4 ± 0.15c 16.5 ± 0.20c 15.3 ± 0.20c 12.6 ± 0.15c 16.2 ± 0.05c

MeOH 19.4 ± 0.15b 18.4 ± 0.05b 17.3 ± 0.15 b 16.1 ± 0.15b 14.7 ± 0.20b 18.2 ± 0.10b

AcN 21.3 ± 0.15a 19.3 ± 0.15a 19.7 ± 0.15a 17.5 ± 0.20a 15.7 ± 0.05a 19.3 ± 0.15a



73

Table 4.6: Minimum inhibitory concentration of PoPx for various gram positive and gram negative microorganisms

PoPx Minimum Inhibitory Concentration (mg/mL ± SEM)

B. subtilis E. coli S. aureus P. aeruginosa S. typhimurium A. niger

Aqueous 0.43±0.02a 0.84±0.05a 0.66±0.05a 0.72±0.05a 1.30±0.09a 1.19±0.05a

EtOH 0.35±0.04b 0.52±0.05bc 0.45±0.04b 0.57±0.03b 0.83±0.03b 0.89±0.04b

MeOH 0.31±0.03b 0.54±0.04b 0.42±0.04bc 0.48±0.03c 0.78±0.02b 0.69±0.03c

AcN 0.25±0.03c 0.46±0.03c 0.36±0.03c 0.41±0.03d 0.66±0.04c 0.55±0.03d

Values sharing same letters are non- significant at p<0.05; SEM = Standard Error of Means


74

4.5 Cytotoxic effects of PoPx in brine shrimps

The brine shrimps lethality assay is generally considered as an inexpensive, rapid and

reliable technique to evaluate cytotoxic effects of drugs and plant extracts. Hydroalcoholic

extracts of PoP at various concentration (10,100 and 1000µg/mL) were evaluated for their

potential cytotoxic effects against freshly grown nauplii. The LC50 of all extracts against

brine shrimps larvae was recorded as >1000 µg/mL indicating PoPx as non-toxic at highest

tested concentration (Table 4.7). On account of its lethality, maximum larvicidal activity

(30%) was observed in methanolic extracts at 1000 µg/mL whereas ethanolic, acetone and

water extracts were observed to have 20, 16.7 and 3.3% larval mortality at the same

concentration, respectively.

PoPx from various solvents were found non cytotoxic at 10 µg/mL and 100 µg/mL.

The results of our study are in line with those reported by Manasathien et al. (2012)

indicating the LC50 of ethanol and water extracts of PoP as 1206.98 and 1743.3 µg/mL,

respectively. One similar study on toxicity from apple juice containing 10mg/mL PoPx

showed a little cytotoxic effect of PoPx at concentration of ~ 10g L-1 juice (Altunkaya et al.

2013b). The low toxicity of PoPx as observed in the current study, corroborated the

foregoing investigations suggesting a safe application of PoPx in potential drugs

formulations, nutraceuticals and functional foods in order to suppress natural or induced

oxidative stress.

75

Table 4.7: Brine shrimps lethality test for pomegranate peel aqueous, ethanolic, methanolic and acetone extracts

PoPx No of shrimps No. of survivors at various concentration of pomegranate

peel extracts

LD 50 (Std drug

Etoposide)

10µg/mL 100µg/mL 1000µg/mL

Aqueous 30 30 30 29 7.46

EtOH 30 30 30 21 7.46

MeOH 30 30 27 24 7.46

AcN 30 29 28 25 7.46

Values are the mean of replicates (n = 3)


76

4.6 Nutritional composition of pomegranate peel and peel supplemented cookies

4.6.1 Nutritional profile of PoP and PoP supplemented cookies

Pomegranate peel in the form of its extracts or as powder may be consumed as

advantage for their health promoting features in the form of nutraceuticals or functional food

ingredients. Bakery products on account of their wide acceptability as consumable good may

serve as most efficacious tool for nutritional enrichment, supplementation and fortification.

In the present study, PoP was supplemented to wheat flour based cookies with the aim to

improve nutritional quality and shelf life stability of the finished product.

Data pertaining to nutritional features of PoP, and PoP supplemented cookies are

presented in Table (4.8). Results revealed an attractive nutritional profile of pomegranate

peel particularly the fiber and inorganic residues of this recyclable fruit waste that might

significantly anticipate their role in reducing caloric contents and improving mineral profile

of the cookies. Relatively slight but non-significant changes in moisture contents of control

and supplemented cookies were observed. A slight reduction in moisture contents of

supplemented cookies was noticed that might be associated with poor water binding capacity

of pomegranate peel as compared to wheat flour.

Results with respect to protein and fat concentrations in PoP revealed pomegranate

peel to be almost devoid of these food fractions. Hence a gradual and significant reduction

pattern in protein contents of cookies was observed i.e. 6.77 (T0) to 6.16% (T5). Reduction in

protein contents of supplemented cookies has also been reported by Nazni et al. (2010)

where protein contents of potato and ragi supplemented biscuits were found to be relatively

lower (7-8%) as compared to wheat flour cookies. However, reduction in protein contents

of cookies associated with PoP supplementation has been reported to have a little impact on

overall availability of protein if augmented with protein from some edible plant sources.

Presently, as has been reproduced from this research, 100g supplemented cookies might

contribute a significant part of protein daily dietary allowance i.e. 0.8g/kg/d (FAO, 2007).

Crude fiber contents of cookies prepared with 100% straight grade flour (SGF) were

found to be six times lower as compared to the cookies prepared with 7.5% PoP

supplementation. Interestingly, on an average daily dietary fiber intake scale i.e.

~11g/1000Kcal, cookies prepared with 100% SGF contribute only ~0.7g of the fiber as

77

compared to cookies supplemented with 7.5% PoP that can provide significantly higher fiber

contents at the same caloric level.

A visible increment in crude fiber contents (80%) of control could be solely

attributed to fiber enriched profile of PoP i.e. 17.53±0.74%.Good inorganic residual pool

(2.7%) was recorded in PoP as compared to control (0.47%). Incorporation of PoP powder in

SGF presented a slight but non-significant (p> 0.05) increment in ash contents of cookies

from 0.53 – 0.76% thereby improving micro-elemental concentration of baked good. PoP

supplementation @ 7.5% increased ash levels of cookies upto 43.4%. Similar results were

presented by Al-Sayed and Ahmed (2013) who reported a significant increase (42.5%) in ash

contents of cakes supplemented with watermelon rind powder at the same replacement level

Higher dietary fiber and low fat profile of PoP supplemented cookies establish them to be a

low caloric food that could be exploited by the people being on restricted diets. Likewise,

utilization of organoleptically acceptable and nutritionally enriched cookies supplemented

with citrus peel were reported by Youssef and Mousa (2012)

78

Table 4.8: Nutritional composition of pomegranate peel, Straight grade flour and pomegranate peel supplemented cookies

(g/100g)

Sample Moisture Ash Protein Fiber Fat Carbohydrates† Caloric Value

(Kcal/100g)

Peel 9.34±0.13b 2.70±0.23a 0.70±0.03h 17.53±0.74a 0.4±0.03f 78.67±0.32b 321.09±0.14h

SGF 12.86±0.21a 0.47±0.01e 12.34±0.04a 0.33±0.03g 1.72±0.1e 85.14±0.18a 405.42±0.09g

T0 4.35±0.01c 0.53±0.01de 6.77±0.06b 0.32±0.02g 23.78±0.04a 68.60±0.02c 515.51±0.21a

T1 4.30±0.02cd 0.55±0.02de 6.63±0.07c 0.68±0.06f 23.64±0.03ab 68.50±0.02c 513.34±0.31b

T2 4.24±0.03de 0.61±0.03cd 6.53±0.07d 1.12±0.05e 23.59±0.02bc 68.15±0.01d 511.02±0.33c

T3 4.17±0.02ef 0.67±0.02bc 6.42±0.08e 1.42±0.06d 23.51±0.01bc 67.98±0.02d 509.14±0.08d

T4 4.13±0.03fg 0.72±0.02b 6.27±0.06f 1.72±0.04c 23.42±0.03cd 67.87±0.01d 507.3±0.24e

T5 4.09±0.03g 0.76±0.01de 6.16±0.10g 1.96±0.06b 23.33±0.04d 67.79±0.03d 505.72±0.07f

Values with same lettering in same column are non significant (p>0.05)

T0= 100% SGF; T1 = 98.5% SGF + 1.5% PoP; T2 = 97% SGF + 3% PoP; T3 = 95.5% SGF = 4.5% PoP; T4 = 94% SGF + 6% PoP; T5 = 92.5% SGF + 7.5%

PoP

†Calculated on dry weight basis as 100 - (Ash+Protein+Fiber+Fat)

79

4.6.2 Mineral profiling of PoP and PoP supplemented cookies

Foods with adequate fiber, minerals, vitamins and other macronutrients significantly

contribute in reducing cardiovascular disorders and associated mortality rates. Dietary

deficiency of micronutrients specifically calcium and potassium have been reported to be

highly associated with the development of cardiovascular disorders (McCarron and Reusser

2001). PoP of Alipuri cultivar was found to hold tremendous concentration of calcium

(1192mg/Kg) and potassium (2749.45mg/Kg) contents (Table4.9). Cookies supplemented

with PoP depicted a significant improvement in calcium and potassium contents of these

cookies. Naturally enriched micro-elemental pool of PoP was found to have improved

calcium and potassium concentration of control by 46.89 - 175.41mg/Kg and 327.26 –

598.61mg/Kg, respectively at 7.5% replacement level i.e. addition of PoP in SGF (Table 4.9).

Micro-elemental profiling of PoP revealed this waste fraction to be containing

1.21mg/Kg of Fe and (3.678mg/Kg of Zn). Present study revealed PoP supplementation to

be a viable approach that has characteristically improved Fe and Zn contents of 100% SGF

cookies from 0.463 – 0.556mg/Kg and 2.645 – 2.769mg/Kg, respectively A non-significant

(p>0.05) effect of PoP substitution was measured in Mn contents of cookies (Table 4.9).

Monotonous diets of impoverished peoples in developing countries are assumed to be

the cofactor in inadequacy of essential minerals. PoP, a non-edible part of pomegranate has

been previously referred as a good source of Ca, K, Mg, Fe and Zn (Fawole and Opara

2012). As stated earlier, on daily dietary scale (FAO 2007), 100g serving of PoP

supplemented cookies could provide better micronutrients levels as compared to normal

wheat flour cookies. Inter comparison of control and supplemented cookies indicated

approximately 270, 82, 16 and 5% higher Ca, K, Fe and Zn concentration from PoP

supplemented cookies respectively. Dietary diversification, supplementation and

incorporation of mineral rich plant material such as PoP in food preparations might serve as a

convenient solution to minerals inadequacy problem of economically distressed populations

(Prentice and Bates 1993; Weaver et al. 1999)

80

Table 4.9: Mineral composition of pomegranate peel and peel supplemented cookies (mgKg-1)

Treatment Ca Na K Mn Cu Fe Zn

Peel 1192.039±2.7a 592.944±1.38g 2749.455±3.78a 0.021±0.02NS 0.021±0.002a 1.210±0.02a 3.678±0.09a

T0 46.895±1.04g 770.909±0.95a 327.261±2.47g 0.017±0.01 0.009±0.002b 0.463±0.02e 2.645±0.08f

T1 79.545±0.83f 765.422±2.51b 382.245±1.14f 0.017±0.03 0.009±0.001b 0.478±0.04e 2.669±0.1ef

T2 104.701±0.71e 758.859±1.53c 444.035±2.01e 0.017±0.01 0.01±0.002b 0.497±0.03de 2.691±0.07de

T3 127.781±0.91d 753.999±1.29d 495.763±0.94d 0.018±0.02 0.01±0.002b 0.514±0.05cd 2.719±0.09cd

T4 149.538±1.43c 749.069±0.83e 545.633±1.38c 0.018±0.01 0.0100.001b 0.536±0.06bc 2.746±0.1bc

T5 175.411±0.48b 743.839±0.58f 598.609±1.62b 0.018±0.01 0.011±0.002b 0.556±0.07b 2.769±0.08b



PoP

Table 4.10: Instrumental parameters for determination of micro elements with atomic absorption spectroscopy

Element Wave

Length (nm)

Lamp

Current

(mA)

Fuel

(Acetylene

flow) L/min

Burner

Height

(mm)

Band Pass

(nm)

Nebulizer

Uptake Time

(Sec)

Excess

curvature

Limit (%)

Detection

Limit (ppm)

Fe 248.3 15 0.9 7 0.2 4 10-40% 0.05

Zn 213.9 12 1.2 7 0.2 4 10-40% 0.01

Mn 279.5 15 1.0 7 0.2 4 10-40% 0.02

Cu 324.8 6 1.1 7 0.5 4 10-40% 0.03

81

4.6.3 Total phenolics and antioxidant capacity of PoP supplemented cookies

Higher concentration of total phenolics (1387mgGAE/100g) and associated DPPH

and ferric reducing antioxidant capacity (87.4% and 275mmol/100g) were exhibited by PoP

(Table 4.11). Total phenolic contents of cookies increased from 90.7mg/100g to

161.9mg/100g suggesting a linear trend with gradual increments of PoP concentration in

SGF i.e. from 0 – 7.5%.

A dose dependent response was observed in antioxidant activity of supplemented

cookies suggesting a correlation among antioxidant activity and total phenolics. At highest

level of PoP supplementation, cookies exhibited ~ 50% DPPH radical scavenging activity.

However, cookies prepared with 100% SGF were attributed with least DPPH radicals

scavenging activity. A linear trend was also noticed from ferric reducing antioxidant power

assay where free radical scavenging value was increased with increasing PoP

supplementation i.e. 0.4 – 20.7mmol/100g. Diversified nature of PoP phenolics on account of

their ability to donate hydrogen atom is one of the reported reason for high free radical

scavenging properties of PoP and its extracts. Correlation between phenolics concentration

and antioxidant activity has been previously confirmed by some research reports

(Chidambara Murthy et al. 2002; Zahin et al. 2010).

Presence of an appreciable concentration of phenolics in PoP supplemented cookies

might render a disease preventive role in addition to food stabilization features. Several

groups of researchers have reported lower phenolics profile (~14.4mgGAE/100g) and

decreased antioxidant activity (~28%) of white flour used in cookies and bread suggesting

an enormous antioxidant potential of PoP when used as a supplement in cookies (Han and

Koh, 2011; Vaher et al. 2010).

Our findings from the current report suggest complimentary effect of PoP on

nutritional and nutraceutical properties of PoP supplemented cookies in terms of improved

phenolics and antioxidant profile. Least concentration of condensed tannins i.e.

proanthocyanidins identified in this research could hardly anticipate toxicity on account of

their possible nutrients absorption and mutagenic properties. The data available on biological

safety of PoP suggest 1420mg of PoP extracts to be non-toxic and safe for consumption as

food or feed ingredient (Heber et al. 2007; Saad et al. 2012).

82

This particular report confirmed PoP supplemented cookies to carry considerably

lower extracts amounts than stated in previous reports on PoP that further endeavor PoP as

non-toxic at fairly acceptable level of supplementation. In order to make PoP

supplementation more practical and adoptable in bakery goods, a complete picture of PoP

supplementation levels and their effect on product overall organoleptic acceptability has been

deliberated in forth coming section.

83

Table 4.11: Total Phenolic Contents and antioxidant activity of pomegranate peel and peel powder supplemented cookies

Treatments Total Phenolic Contents

(mgGAE/100g)

Antioxidant Activity

DPPH (%) FRAP mmol/100g

PP 1387±3.04a 87.4±2.30a 275±1.34a

T0 90.7±1.98g 27.3±1.86e 0.39±0.03e

T1 104.57±2.25f 33.57±1.88d 4.47±.010de

T2 119.1±2.01e 37.57±2.25d 8.67±0.09cd

T3 133.9±2.20d 43.17±2.00c 12.67±0.24c

T4 147.3±1.55c 46.47±1.60bc 16.85±0.16b

T5 161.87±1.86b 49.37±2.12b 20.7±0.81b



PoP

84

4.7 Cookies stability study - free fatty acid levels

Cookies are often exposed to quality degradation due to moisture loss, oxidation,

textural alteration and spoilage during prolonged storage (Adegoke et al. 1998). Owing to

higher fat contents, cookies are at higher risk of oxidative changes. Addition of natural

ingredients holding antioxidant properties or synthetic antioxidant prevent nutritive losses by

retarding or inhibiting oxidation reactions (Reddy et al. 2005). However, synthetic

antioxidants have been reported as controversial with respect to their safety for utilization in

food products (Nanditha and Prabhasankar 2008).

PoP is considered as a plentiful source of phytochemicals more specifically

ellagitannins that impart characteristic free radical scavenging properties. Development of

off-flavors during storage is the indicative of product oxidation that can be identified by

measuring free fatty acids (FFA) levels. Findings from the current research work validate

PoP phenolics to offer a significant role in inhibiting oxidation of PoP supplemented cookies

during storage as compared to the normal cookies (Table 4.12).

Higher levels of FFA (0.399%) were observed in control (T0) samples after 04months

storage as compared to T5 samples where FFA level were recorded to be 0.195%. Similar

results were reported by Maisuthisakul et al. (2007) who suggested plant phytochemicals to

be contributing higher antioxidant capacity as compared to some normal controls such as α –

tocopherols in crackers stabilization. Relatively higher but permissible FFA levels (0.82 –

1.20%) in cookies and crackers have also been reported by Daglioglu et al. (2004) in a

twelve-month storage study.

85

Table 4.12: Free fatty acid (%) levels in pomegranate peel supplemented cookies stored for a period of 4months

Sample Initial Reading 2nd Month 4th Month

T0 0.183±0.01a 0.22±0.02a 0.399±0.01a

T1 0.164±0.01ab 0.195±0.01ab 0.353±0.01ab

T2 0.138b±0.01c 0.169±0.01abc 0.292±0.02bc

T3 0.118±0.01c 0.138±0.01bc 0.23±0.01c

T4 0.065±0.02d 0.128±0.02bc 0.22±0.01c

T5 0.077±0.01d 0.113±0.02c 0.195±0.01c



PoP

86

4.8 Organoleptic evaluation of PoP supplemented cookies

Supplementation of wheat flour with relatively inexpensive edible plant materials

helps in improving nutritional quality of the wheat based eatables. However; structural

deformities and loss of sensorial features in supplemented foods are a few drawbacks of

supplementation that if not addressed adequately, might significantly affect consumer

acceptance and product marketability. Organoleptic evaluation therefore provides very

important information on food quality critically analyzing human response towards food

quality attributes i.e. taste, smell, color, texture and other sensory features.

Organoleptic evaluation of PoP supplemented cookies indicated a significant effect

(p<0.05) of supplementation on cookies color (Table 4.13). Highest color acceptability score

was observed in control followed by cookies with 2.5% peel powder supplementation.

Although consumer score was markedly declined with increasing supplementation levels of

PoP however the product remained in acceptable range (6.16) at 9point hedonic scale (Table

4.13). Pomegranate peel powder contains higher ellagitannins fractions which was evident

from colored suspension, imparting characteristic yellow color to the finished goods. A

similar trend was recorded by Turksoy and Ozkaya (2011) reporting significant reduction in

color scores of cookies supplemented with β-carotene rich pumpkin peel powder and carrot

pomace powder.

PoP supplemented cookies were not found highly acceptable at supplementation level

of 7.5%. In the course of sensory evaluation of PoP supplemented cookies, amongst all

sensory parameters, taste of the product remained below acceptability range at 7.5%

supplementation level however, slight to moderate acceptability scoring was attributed by the

panelist at 2.5 – 6.0% supplementation. In order to improve acceptability and consumption

levels of astringent functional food preparations, bitter taste blockers possessing human taste

receptors antagonistic properties have been identified as ideal solution to organoleptic

concerns (Bom et al. 2012; Karanewsky et al. 2012).

A significant decline in score for crispiness of 100% SGF cookies on PoP

supplementation was recorded i.e. 7.44 – 6.13 that might be associated with increase in fiber

contents of cookies. Fiber mediated tenderness and moistness in fiber supplemented cookies

87

have been previously reported by Jeltema et al. (1983). Textural hardness features of cookies

have also been reported to be associated with fiber contents (Uysal et al. 2007).

As has been witnessed from the nutritional composition of PoP supplemented

cookies, PoP supplementation @ 7.5% gradually improved fiber contents of cookies from

0.32 – 1.96% that might depict product hardening property as has been noticed from the

characteristic sensorial score decline at maximum level of supplementation i.e. 7.5% (Table

4.13). PoP supplementation as indicated in organoleptic study data resulted in reduction in

organoleptic acceptability as compared to 100% wheat flour control cookies. However,

overall acceptability scores of cookies made with ~ 6.0% PoP supplementation level were

noticed indicating PoP supplementation at this level to be feasible strategy for utilization of

this fruit waste in food products.

88

Table 4.13: Quality scores of cookies supplemented with different levels of pomegranate peel

Treatment Taste Color Crispiness Texture Overall Acceptability

T0 7.36±0.21 a 7.4±0.05a 7.4±0.07a 7.3±0.07a 7.42±0.02a

T1 6.58±0.07b 6.620.02b 6.68±0.04b 6.52±0.02b 6.72±0.04b

T2 6.04±0.03bc 6.56±0.02b 6.28±0.03bc 6.16±0.04b 6.26±0.02bc

T3 6.48±0.05bc 6.58±0.01b 6.16±0.03bc 6.26±0.02b 6.24±0.02bc

T4 5.84±0.13c 6.46±0.03b 5.92±0.05c 6.18±0.01b 5.94±0.03c

T5 6.28±0.03bc 6.26±0.02b 6.16±0.02bc 6.14±0.1b 6.24±0.01bc



PoP

89

4.9 Effect of supplementing PoPx and PM in Cookies

4.9.1 Nutritional composition of PoPx and PM supplemented cookies

Given the significant contribution of cereals to meet daily caloric requirement in

many parts of the world yet these are not considered complete and balanced source of highly

important class of micro and macronutrients. This situation has lead the scientific community

and program managers to follow fortification and supplementation programs. Bakery

products specifically cookies, supplemented with nutraceuticals like polyphenols and dietary

fibers yield a wide range of health benefits to the consumers.

The upshots of the present research endeavor revealed PM and PoPx supplemented

cookies to contain substantially increased dietary fibers and inorganic residues. PM

supplementation at 7.5% in straight grade flour improved cookies fiber and ash contents by

65% and 18%, respectively (Table 4.14).

PoP supplementation improved electrolytes profile of cookies suggesting PM

supplemented cookies to be the better source of Na, Ca and K in addition to increased fiber

concentration. PM, being very low in fat and protein, as supplement did not significantly

increase the caloric value of cookies i.e. 500.9Kcal (100g-1) in T5 to 508.01Kcal (100g-1) in

T0. Contrarily, PoPx being rich in phenolics and devoid of fat, protein and carbohydrates

were found to elicit non-significant effect of supplementation on nutritional composition of

cookies. The validity of our findings is supported by a recent study by Bertagnolli et

al.(2014) who reported guava peel supplementation to increase cookies fiber contents by

15.2% suggesting fruit peels to be a potential ingredient in manufacturing fiber dense food

products.

PoPx and PM supplementation might therefore be considered as a novel approach to

bring nutritionally rich underutilized fruit waste in food circle therefore our findings seem to

be promising and match those of Mildner-Szkudlarz et al. (2013) who reported pomace of

white grapes to possess the ability to increase cookies fiber pool by 88% at 10%

supplementation level. High caloric foods with lower protein concentration are generally

recommended in renal failure patients (Mochizuki et al. 2000). PM, being extremely low in

protein and rich in carbohydrates can also be tested for supplementation at higher levels in

protein devoid caloric dense foods.

90

Table 4.14: Nutritional composition of PoPx and PM supplemented cookies g(100g)-1

Values bearing different lettering in the same column are significantly (p<0.05) different from each other; Mean ± SD

PM= Pomegranate peel bagasse

SGF= Straight grade flour T0= 100% straight grade wheat flour

T1=1.5% PM+ 98.5% SGF, T2=3.0% PM+ 97.0% SGF, T3=4.5% PM+94.5% SGF, T4=6.0% PM+ 94.0% SGF, T5=7.5% PM+ 92.5% SGF, T6=0.25%

Pomegranate peel extracts (PoPx) + 99.75% SGF , T7=0. 50% PoPx+99.5% SGF, T8=1.00% PoPx+ 99.0% SGF.

Sample Moisture Ash Protein Fiber Fat Carbohydrates Caloric value

(Kcal/100g)

Pbg 4.32±0.06 2.74±0.07 0.64±0.05 16.77±0.21 0.21±0.03 79.64±0.20 323.03±1.19

SGF 12.60±0.26 0.97±0.04 13.14±0.19 0.56±0.05 1.61±0.06 83.72±0.17 401.92±0.35

T0 3.72±0.04a 1.00±0.05e 6.97±0.02a 0.57±0.03gh 22.86±0.47a 68.61±0.02c 508.01±0.26a

T1 3.61±0.04b 1.06±0.02d 6.86±0.02bc 0.81±0.04e 22.74±050c 68.53±0.01d 506.23±0.19b

T2 3.53±0.04bc 1.08±0.03cd 6.81±0.03c 0.99±0.06d 22.67±0.34d 68.46±0.03e 505.01±0.25c

T3 3.45±0.04cd 1.11±0.03bc 6.72±0.02d 1.21±0.04c 22.60±0.23e 68.37±0.02f 503.71±0.22d

T4 3.32±0.03ef 1.14±0.05ab 6.64±0.04e 1.44±0.03b 22.51±0.20f 68.28±0.03g 502.24±0.18e

T5 3.26±0.03f 1.18±0.04a 6.53±0.03f 1.62±0.04a 22.42±0.15g 68.25±0.01g 500.91±0.24f

T6 3.37±0.04fde 0.93±0.04f 6.90±0.02ab 0.57±0.03h 22.81±0.29ab 68.79±0.04b 508.11±0.07a

T7 3.48±0.03c 0.90±0.04fg 6.88±0.04b 0.59±0.03fg 22.79±0.32bc 68.84±0.05ab 508.00±0.07a

T8 3.61±0.04b 0.87±0.04g 6.86±0.03bc 0.60±0.03f 22.77±0.31bc 68.90±0.03a 507.99±0.10a

91

4.9.2 Total phenolics and antioxidant potential of cookies

Wheat, as a staple food crop for a number of international communities is normally

considered low in poly phenols i.e. 0.02 – 0.04% (Theuer 2002), thereby representing poor

bioactive contents in SGF. Addition of plant extracts in SGF, is intended to impart functional

characteristics to the finished products, which not only build total phenolics levels but also

enhances antioxidant properties of the consumable products.

Table (4.15) demonstrates gradual increase in total phenolics concentration in PM and

PoPx supplemented cookies. PM being low in phenolics, presented relatively lower free

radical scavenging properties i.e. 78.35 – 92.91 mgGAE(100g)-1 as compared to PoPx

(123.61 – 315.70mgGAE(100g)-1). Highest phenolics concentration was noticed in T8

whereas minimum levels of phenolics were noted in T0, indicating PoPx supplementation in

SGF by 1.0% to increase total phenolics contents of the cookies by 76%. PoPx and PM

supplemented cookies presented significant antioxidant potential as well (Table 4.15) A

positive correlation was observed for PM with peel phenolics and free radical scavenging

activity i.e. r2 = 0.98 for total phenolics and DPPH and r2 = 0.99 for total phenolics and

FRAP. Antioxidant activity of cookies supplemented with PM and PoPx indicated gradual

built in product’s phenolics pool and significantly (p<0.05) increased FRAP and DPPH free

radical scavenging properties (Table 4.15).

Our findings further validated previous reports suggesting fruit peels, on

supplementation to bakery products, held the potential to establish a positive correlation

between concentration of supplementary material, dietary fibers, total phenolics and

antioxidant capacity of the finished goods (Rupasinghe et al. 2008). Interestingly higher free

radicals scavenging properties were imparted by PoPx supplementation as compared to PM.

Cookies produced from SGF, supplemented with 1.0% extracts, increased FRAP from 1.25

to 58.75 while that of DPPH from 22.14 to 64.83% mmol (100g)-1.

92

Table 4.15: Total phenolics and antioxidant profile of pomegranate peel meal and extracts supplemented cookies


PM= Pomegranate peel meal


T1=1.5% PM+ 98.5% SGF, T2=3.0% PM + 97.0% SGF, T3=4.5% PM +94.5% SGF, T4=6.0% PM + 94.0% SGF, T5=7.5% PM + 92.5% SGF, T6=0.25%


Treatments

Total phenolic contents

mgGAE/100g

Antioxidant properties

FRAP

(mmol/100g)

DPPH

(%RSA)

T0 75.19±0.21i 1.25±0.04g 22.14±0.24i

T1 78.35±0.29h 1.51±0.03g 23.21±0.31h

T2 82.47±0.29g 1.65±0.04g 24.80±0.32g

T3 87.53±0.41f 2.50±0.08f 26.66±0.31f

T4 90.38±0.46e 3.75±0.07e 27.99±0.46e

T5 92.91±0.43d 4.50±0.14d 29.19±0.29d

T6 123.61±0.99c 20.25±0.56c 37.30±0.66c

T7 205.89±0.89b 32.50±0.58b 48.60±0.44b

T8 315.70±0.87a 58.75±0.41a 64.83±0.42a

93

Cookies supplemented with PM(1.5% to 7.5%) depicted weaker free radicals

scavenging properties as compared to those treatments where supplementation was carried

with various levels of PoPx i.e. 0.25 – 1.0% (Table 4.14). Since, PM supplemented wheat

flour has been shown to hold a reasonable phenolics pool and antioxidant properties as

compared to the control therefore, consumption of PoPx supplemented cookies could be

exploited as nutritionally viable and potential strategy to enhance nutraceuticals properties of

the consumable products.

4.10 Microbiological stability of cookies

Microbiological safety of the bakery products is not considered a matter of concern

because baking is generally carried out at extremely higher temperature. The baking

primarily destroys potential pathogens and minimizes microbial food spoilage factors.

However baking at lower temperature, mishandling during packaging, cross contamination

and improper food storage sound to be the potential factors that may negatively alter the

safety and quality status of the baked goods (Knight et al. 1961; Saranraj 2012).

The data presented in Table 4.16, indicate least to no observation of total bacterial

count on growth medium plates inoculated with PM and PoPx supplemented cookies

samples. A non-significant difference of total bacterial counts and yeasts / molds was

recorded in cookies supplemented with 1.5 to 4.5% PM whereas a significant reduction in

microbiological load was noticed in the treatments carrying 6.0 - 7.5% PM and 0.25 - 1.0%

PoPx (Table 4.16). Comparing with the control, PoPx supplemented cookies showed

relatively controlled microbiological stability during prolonged storage for a period of three

months. No visible growth of aerobic plate counts and yeast/moulds was detected in cookies

supplemented with PoPx however; total plate counts and yeast / molds growth was identified

as 1.6 log10cfu/g and 1.17 log10cfu/g, respectively in 100% wheat flour cookies.

94

Table 4.16:Microbiological stability of PoPx and PM supplemented cookies

Treatments Total Plate Counts (log10cfu/g) Yeast/Molds (log10cfu/g)

0 days 30 days 90 days 0 days 30 days 90 days

T0 1.14±0.06a 1.60±0.04a 2.04±0.02a ND 1.17±0.09a 1.70±0.03a

T1 1.13±0.05a 1.54±0.04ab 2.04±0.02a ND 1.08±0.07ab 1.65±0.03ab

T2 0.99±0.09b 1.50±0.05abc 2.04±0.04a ND 0.96±0.06b 1.61±0.05ab

T3 0.99±0.09b 1.46±0.05bc 1.98±0.03ab ND 0.97±0.10b 1.54±0.08bc

T4 ND 1.39±0.08cd 1.90±0.03bc ND ND 1.44±0.04cd

T5 ND 1.30±0.09d 1.84±0.03c ND ND 1.39±0.05d

T6 ND 1.15±0.13e 1.60±0.05d ND 1.01±0.11ab 1.39±0.09d

T7 ND 1.07±0.11e 1.47±0.07e ND ND 1.26±0.08e

T8 ND ND 1.30±0.09f ND ND 1.05±0.11f






95

Data pertaining to microbial growth in cookies indicated significant (p<0.05)

reduction in total plate counts and yeasts/molds growth rates in PoPx supplemented cookies

as compared to PM and control cookies (Table 4.16). Total plate counts and yeasts / molds of

control samples were found to be ranging from 1.14 to 2.04 log10cfu/g and <10cfuto 1.70

log10cfu/g, respectively during 0-90 days storage at ambient temperature. At variance with

the control, PM carrying residual levels of PoP phenolics presented significant inhibition in

microbial growth during storage for 90days. Cookies supplemented with 7.5% PM showed

relatively lower level of total plate counts and yeasts / molds i.e. 1.84 log10cfu/g and 1.39

log10cfu/g, respectively after 90 days storage.

Our results showed PoPx, to exert increased antimicrobial properties in the finished

good as compared to previously reported results (Saeed et al. 2013) where researchers

demonstrated 2.5% supplementation of clove extracts in bread to be inhibiting aerobic plate

counts by 11.4%. The reported level of inhibition was primarily a proxy of increased ability

of PoPx to act as a natural antimicrobial in food systems as compared to their available

counterparts i.e. PM.

4.11 Oxidative stability of cookies

4.11.1 Thiobarbituric acid contents of PoPx and PM supplemented cookies

Oxidative instability has been shown to exist in cookies for being rich in lipids

contents. Extremely higher baking temperature and lipids hydrolytic degradation during

inappropriate storage are predominant precursors to oxidative instability of the baked

products. PoPx featured with vital phenolics profile imparts characteristic inhibition of lipid

oxidation in food systems therefore PoPx has gained popularity as natural preservative in

high lipid carrying food preparations. Levels of thiobarbituric acid – a lipid oxidation marker,

were measured in PoPx and PM supplemented cookies (Table 4.17).

The study delineated significant (p<0.05) reduction in Thiobarbituric acid reactive

substances (TBARS) value of cookies supplemented with 0.25 – 1.0% PoPx i.e. 0.642% in

control to 0.209 % in T8. The 90days ambient temperature storage study of PM and PoPx

supplemented cookies revealed significant preventive role of PoPx in inhibiting oxidative

deterioration of lipids as compared to the cookies made with 100% wheat flour. The results

of the present study presented in Table 4.17 reveal T8 (1.0% PoPx supplemented) to be the

96

best treatment in terms of developing decreased TBARS levels (2.619%) in comparison

with those developed by control treatment (3.792%) after 90 days storage

4.11.2 Effect of PoPx and PM supplementation on free fatty acids contents of cookies

Deep frying or accelerated heating of lipids are sometime desirable to enhance food

sensory properties however; thermal abuses of lipids are reportedly associated with escalated

levels of lipid oxidation indicators predominately peroxides and free fatty acids (FFA) (Tyagi

and Vasishtha 1996).

In addition to their ability to inhibit TBARS production, PoP extracts (0.25 – 1.0%)

exhibited better stability of PoPx supplemented cookies towards lipid oxidation. Cookies

prepared with 1.0% PoPx showed significantly lower levels of FFA (0.10%) as compared to

the control treatment (0.19%) (Table 4.18). Better lipid oxidation inhibition rate was

recognized in PoPx supplemented cookies followed by cookies carrying 7.5% PM. Non-

significant effect of PM supplementation on inhibition of FFA development was observed

and increase in lipids deterioration was almost parallel to the one observed in 100% wheat

flour cookies. Our work confirmed the results of the recent study on lipid oxidation

inhibition in meat balls treated with 0.1 – 0.3 % crude extracts of PoP (Ozdemir et al. 2014).

97

Table 4.17: Thiobarbituric acid contents (mgMDA(Kg)-1) of PoPx and PM supplemented cookies

Treatments TBA (mgMDA/Kg)

0 Days 30days 90days

T0 0.642±0.029a 2.322±0.049a 3.792±0.044a

T1 0.565±0.047ab 2.144±0.036b 3.708±0.047ab

T2 0.557±0.054 ab 2.027±0.048b 3.613±0.034bc

T3 0.545±0.043 ab 1.851±0.037c 3.534±0.049cd

T4 0.537±0.052 ab 1.762±0.066cd 3.477±0.041cd

T5 0.529±0.038 ab 1.689±0.034d 3.404±0.054d

T6 0.484±0.029bc 1.165±0.067e 3.027±0.057e

T7 0.397±0.037c 0.982±0.024f 2.839±0.056f

T8 0.209±0.010d 0.618±0.013g 2.619±0.014g






98

Table 4.18: Effect of PoPx and PM supplementation and storage on free fatty acids levels of cookies (%)

Treatment FFA (%)

0 days 30 days 90 days

T0 0.19±0.01a 0.48±0.07a 0.55±0.07a

T1 0.18±0.04ab 0.45±0.06ab 0.56±0.07ab

T2 0.18±ab0.03 0.44±0.06ab 0.66±0.09ab

T3 0.17±0.03abc 0.42±0.05ab 0.57±0.07ab

T4 0.17±0.02abc 0.42±0.04abc 0.52±0.06b

T5 0.16±0.02abc 0.41±0.04abc 0.51±0.07bc

T6 0.13±0.02abc 0.34±0.05bc 0.38±0.05cd

T7 0.11±0.01bc 0.31±0.04bc 0.36±0.05d

T8 0.10±0.01c 0.28±0.04c 0.34±0.05d






99

Relatively higher lipid oxidation inhibition rates i.e. upto 41% in term of TBARS

number were observed by Devatkal et al. (2014) in PoPx augmented goat nuggets. Current

study, even though exhibited lower inhibition rate of PoPx than reported by Devatkal et al.

(2014) however; product has shown to possess better stability in 90 days storage as compared

to 25 days storage as reported by those researchers. Our work therefore suggests PoPx to

hold significant potential for reducing oxidative stress in low moisture and lipid rich food

products.

4.12 Organoleptic acceptability of PM and PoPx supplemented cookies

Data pertaining to organoleptic properties of PM and PoPx supplemented cookies are

presented in Tables 4.18 and 4.19. As mentioned earlier, PoP supplementation @ 7.5%

imparted significant bitterness to the cookies and was scored least on nine point hedonic

scale for organoleptic acceptability. Contrarily, higher level of sensory acceptability was

identified from cookies supplemented with PoPx @1.0% and PM (7.5%) and none of the

treatments was ranked below the threshold level i.e. sensory score between 5 – 6 for taste,

color, crispiness, texture and overall acceptability.

Cookies with 7.5% PM were graded lower for texture and crispiness after third month

of storage. Reduction in acceptability score for these attributes might be associated to the

improved fiber pool of cookies added vide PM supplementation and its better ability to bind

moisture. Dietary fibers either as component of different flour or in their pure forms i.e.

inulin, were also reported by Popov–Raljic et al. (2013) to be associated with loss in cookies

textural properties during storage.

Cookies supplemented with PoPx were found to be very much compatible to the

control samples (without PM or PoPx) during entire storage period and were liked very much

to moderate with increasing level of supplementation. Organoleptic evaluation of sample

with no supplementation indicated upto 8.3% decline in sensory acceptability while cookies

supplemented with PM and PoPx presented better organoleptic stability as indicated from the

overall acceptability score in 90days storage study. The study indicates maximum level of

PM and PoPx supplementation to have a stable score for overall acceptability i.e. 7.20 and

8.01, respectively at the end of 90days study period. Lipid oxidation and formation of

100

undesirable volatile compounds significantly affected organoleptic acceptability particularly

aroma and taste of high lipid carrying food products (Chemat et al. 2004).

Cookies made with 100% wheat flour, being poor carrier of antioxidants, presented

higher degree of reduction in organoleptic acceptability while PoPx supplemented cookies

being rich in natural free radical scavengers, exhibited better product organoleptic stability

thus suggesting addition of PoPx to cookies as a tool to enhance sensorial properties of the

product. Addition of higher concentration of PM in cookies moderately affected the

product’s color as the finished product was slightly dark in color as compared to the control

sample (Table 4.19 & 4.20).

Astringency attributed to PoP phenolics and flavonoids is one of the most significant

limiting factors in sensory acceptability of PoP supplemented food products. PM being low

in phenolics and flavonoids imparted little or no astringency in the finished product whereas,

caramalization and loss of significant concentration of astringent compound not only reduced

bitterness in PoPx supplemented cookies but also reduced radical scavenging capacity of the

extracts (Table 14) obtained from the source i.e. PoP, bearing ~87% DPPH scavenging

capacity (Ismail et al, 2014). Phytonutrients having ability to deliver functional properties in

disease prevention, cannot be segregated from their sensory such as taste and flavor

properties (Drewnowski 2000). Supplementation of either PoP, PM or PoPx therefore

considers need for communal strategies to improve palatability and sensory properties of the

supplemented products.

101

Table 4.19: Effect of PoPx and PM supplementation and storage on taste and color properties of cookies

Taste Color

Treatment 0 days 30 days 90 days Mean 0 days 30 days 90 days Mean

T0 8.92±0.27a 8.64±0.49a 8.18±0.44bc 8.58a 8.84±0.37a 8.56±0.50a 8.22±0.47ab 8.54a

T1 8.84±0.37a 8.7±0.46a 8.26±0.49b 8.60a 8.58±0.50b 8.52±0.51ab 8.06±0.42b 8.39b

T2 8.5±0.51b 8.4±0.50b 8.06±0.31c 8.32b 8.36±0.49cd 8.2±0.40cd 7.86±0.40c 8.14c

T3 8.12±0.59c 8.04±0.53c 7.84±0.42d 8.00c 8.02±0.51e 7.98±0.47ef 7.78±0.46cd 7.93d

T4 7.74±0.44d 7.7±0.46d 7.54±0.50e 7.66d 7.84±0.37e 7.8±0.40f 7.62±0.49d 7.75e

T5 7.44±0.50e 7.4±0.50e 7.18±0.48f 7.34e 7.54±0.50f 7.48±0.51g 7.26±0.53e 7.43f

T6 8.76±0.43a 8.68±0.47a 8.5±0.51a 8.65 8.5±0.51bc 8.44±0.50ab 8.34±0.48a 8.43a

T7 8.52±0.51b 8.38±0.49b 8.26±0.44b 8.39 8.4±0.50bcd 8.36±0.49bc 8.22±0.46ab 8.33a

T8 8.3±0.51c 8.18±0.48c 8.02±0.32c 8.17 8.22±0.55d 8.14±0.50de 8.06±0.51b 8.14b




T1=1.5% PM + 98.5% SGF, T2=3.0% PM + 97.0% SGF, T3=4.5% PM+94.5% SGF, T4=6.0% PM + 94.0% SGF, T5=7.5% PM + 92.5% SGF, T6=0.25%


102

Table 4.20: Effect of PoPx and PM supplementation and storage on crispiness, texture and overall acceptability of cookies

Crispiness Texture Overall Acceptability

Treatment 0 days 30 days 90 days Mean 0 days 30 days 90 days Mean 0 days 30 days 90 days Mean

T0 8.7±0.46a 8.48±0.51a 8.1±0.42ab 8.43a 8.78±042a 8.4±0.50a 8.14±0.35b 8.44a 8.8±0.40a 8.5±0.51a 8.16±0.42a 8.49a

T1 8.54±0.50ab 8.44±0.54a 7.96±0.53b 8.31b 8.6±0.49ab 8.48±0.50a 8.02±0.43bc 8.37a 8.5±0.51b 8.38±0.49ab 7.92±0.34b 8.27b

T2 8.28±0.45cd 8.1±0.46c 7.74±0.44c 8.04c 8.44±0.50bc 8.24±0.47bc 7.82±0.44d 8.17b 8.3±0.46cd 8.18±0.44cd 7.84±0.51bc 8.11c

T3 7.88±0.59e 7.7±0.46d 7.50±0.51d 7.69d 7.94±0.55e 7.72±0.54d 7.58±0.54e 7.75c 7.88±0.39e 7.82±0.39e 7.68±0.47c 7.79d

T4 7.58±0.50f 7.48±0.50e 7.34±0.48d 7.47e 7.64±0.48f 7.52±0.51e 7.32±0.47f 7.49d 7.72±0.45e 7.62±0.49f 7.44±0.50d 7.59e

T5 7.18±0.48g 7.08±0.44f 6.92±0.44e 7.06f 7.26±0.44g 7.14±0.41f 6.98±0.38g 7.13e 7.34±0.48f 7.2±0.40g 7.06±0.37e 7.20f

T6 8.4±0.50bc 8.34±0.48ab 8.24±0.43a 8.33a 8.48±0.51bc 8.42±0.50ab 8.36±0.53a 8.42a 8.42±0.50bc 8.32±0.47bc 8.24±0.43a 8.33a

T7 8.28±0.45cd 8.16±0.37bc 8.10±0.36ab 8.18b 8.36±0.49cd 8.24±0.43bc 8.08±0.49bc 8.23b 8.24±0.45d 8.16±0.37cd 8.14±0.45a 8.18b

T8 8.16±0.37d 8.06±0.31c 7.96±0.28b 8.06c 8.18±0.44d 8.08±0.34c 7.92±0.34cd 8.06c 8.14±0.37d 8.04±0.28d 7.86±0.35b 8.01c






103

4.13 Safety Evaluation of Pomegranate Peel, extracts and Peel Meal

Functional foods and other novel compounds are thought to play a positive role for

human body. These compounds might carry some toxicity thereby calling for evaluation

before these are permitted for use with regard to human consumption. Efficacy study on

animal models is an appropriate technique to monitor toxicological effects of plant or animal

based biomolecules, preparations and/or food components. Preferences of animal study over

human study is based on account of its easy and safe handling, restricted diet plans,

controlled environmental conditions and close supervision. It is rather difficult to observe

experimental conditions due to high degree of variability in diet consumption patterns and

poor trends to follow diet restrictions plans in human models. Keeping in view the principle

experimental requirements, albino wistar male rats were procured from animal rearing

facility of University of Lahore, Punjab. The study was further divided into three segments

each comprising of three selected treatments to ensure plant material safety at minimum and

maximum acceptable dietary supplementation levels. To our understanding, this study is the

first of its kind that deals with nutritionally and organoleptically acceptable pomegranate

peel, extracts and meal supplementation and their safety as ingredient of choice in ready to

eat food preparations. Efficacy study in this research,basically deals with the effect of

pomegranate peel, their extracts and meal fraction supplementation on animal feed intake,

feed intake associated body parameters, body weight gain, individual organ weight and

serum / blood chemistry. Interactions were studied among controls and other treatments,

treatments and levels of the treatments.

As mentioned in materials & methods, the animals were divided into four groups, the

control and pomegranate peel, peels extracts and peel meal fed rats group. Each group except

the control was further divided into three subgroups each carrying nine animals of similar age

and weight. This section contains results obtained from the efficacy study and their

interpretation.

104

4.14 Effect of PoP, PM and PoPx on feed intake and feeding patterns of rats

Pomegranate peel being an abundant source of high ranking polyphenols particularly

ellagitannins is suspected to impart toxicity on consumption as food ingredient and might

influence feed intake and feeding patterns like weight gain, protein intake, digestibility and

feed conversion efficiency. Feeding diet supplemented with various levels of PoP, PM and

PoPx for a period of eight weeks revealed significant effect on feed intake, weight gain,

protein intake, digestibility and feed conversion efficiency in male rats. Significant

interaction (p<0.05) was observed for treatments and study period except that of protein

intake where non-significant interaction was observed between the assigned parameters

(Table 4.21).

Table 4.21: Mean Square of rats’ diet intake and associated parameters

Source Feed Intake Weight Gain Protein

Intake

FCE Digestibility

Treatment 6.311** 0.738** 0.073** 28.643** 10.200**

Week 346.894** 2.271** 3.620** 3.379** 32.020**

Treatment*week 0.354** 0.021** 0.003ns 0.388** 0.516**

Error 0.127 0.005 0.003 0.059 1.09

** = Highly significant at p<0.05 ns = non-significant (p>0.05)

105

4.14.1 Effect of PoP, PM and PoPx supplementation on feed intake (g) in male albino wistar

rats

Non-conventional dietary sources particularly inedible fractions of fruits and

vegetables are rarely consumed as source of macro or micronutrients due to poor sensory

features. Feeding pomegranate peel and its various fractions viz PoP, PM and PoPx at

various concentrations has been shown to exert a significant effect on feed intake levels

among albino wistar rats (Table 4.21).

Data presented in figures (4.1, 4.2 & 4.3) represent a linear progression in feed intake

(g/rat/day) during entire study period of 56days. Higher feed intake levels were noticed

among rats’ group fed on diet supplemented with PM followed by PoPx whereas, rats’ group

fed on PoP supplemented diet indicated lower feed intake as compared to the control and

treatment groups. Maximum feed intake i.e. 14.5 – 25.355g/day and 14.849 – 24.552g/day

was observed among rats fed on diet supplemented with 2 and 4% PM, respectively. A slight

decline in feed intake was observed among rats fed on the diet with 6% PM supplementation.

Consistent diet consumption pattern was observed among rats fed on diet supplemented with

0.25, 0.50 and 1.0% PoPx and maximum intake was noted at 0.50% supplementation i.e.

24.243g.

Sayed Ahmed (2014) reported a significant reduction in feed intake by rats fed with

pomegranate indicated least feed intake (22.275g) in rats fed on 6% PoP supplemented diets

however the diet consumption rate at 2% supplementation level was found to be comparable

with rats group fed on standard diet. Plant phenolics particularly the tannins are known to

bear significant astringency leading to reduced feed intake levels among rats (Jung et al.

1983). Present study has demonstrated that astringency associated with low and high

molecular weight tannins in PoP might be reduced to some extent by adopting selective

alcoholic extraction techniques as higher feed intake levels were observed in PoPx

(ethanolic extracts) supplemented group.

106

Figure 4.2: Effect of PoP (4.0%), PM (4.0) and PoPx (0.50%) supplementation on feed intake



107

4.14.2 Effect of PoP, PM and PoPx supplementation on weight gain (g) in male albino

wistar rats

Weight gain index in rats (average weight ~144g at 0 day) fed on standard, PoP, PM

and PoPx supplemented diets was calculated and the results were presented in Figure 4.4 –

4.6. These results revealed that the rats groups fed on 2.0% PoP and 0.25% PoPx

supplemented diets did not show significant difference in weight gain when compared with

the rats group fed on standard diet, however rats fed on 2% PM supplemented feed gained

weight significantly higher than the rats in control group at 8th week of feeding.

Weight score for PM supplemented group remained quite stable at 6.0%

supplementation (Figure 4.4). The results manifested a correlation between feed intake and

weight gain among rats fed on PoP supplemented diet as decreased feed intake lead to

reduced weight gain at 4% (1.21 – 1.783g) and 6% (1.17 – 1.73g) supplementation levels.

Available data are also suggestive of the significant role of supplemented plant material in

the feed in attaining weight gain as supplementation levels beyond 2% reduced the feed

intake and weight gain in a dose dependent manner. The findings suggest that PM and PoPx

might be consumed without any risk of significant decline in animal weight at 2.0 – 6.0%

(PM) and 0.25 – 1.0% (PoPx) supplementation respectively. While comparing the effect of

type of supplements on the weight gain among rats present study demonstrated PM to be

holding increased tendency to improve weight with respect to control, PoPx and PoP group

with values being upto 114 g, 112 g, 98 g and 81 g, respectively at 56th day of study. There

have been previous studies comparing the effect of PoP supplementation on weight gain in

rats suggesting an increment in weight to ~ 49 g to the initial weight after 28 days at 10 %

PoP supplementation level (Ashoush et al. 2013).

Contrary to the present findings, lower weight gain index as recorded in rats fed on

PoP and PoPx supplemented diet, shows that waste fraction is capable of reducing weight

and hence may be explored for their ability to be exploited as a tool to control obesity and or

weight management. Lower tendency of rats fed on PoP, its extracts and meal supplemented

diet also indicate PoP and its fraction as a viable ingredient to maintain healthier weight.

108

Figure 4.4: Effect of PoP (2.0%), PM (2.0) and PoPx (0.25%) supplementation on weight gain



109

4.14.3 Effect of PoP, PM and PoPx supplementation on protein intake (g) in male albino

wistar rats

Pomegranate contributes to the total fruit waste to the tune of 30% and has been

regarded as a fruit almost devoid of protein fraction(Ismail et al. 2014). The composition of

the fruit thus suggest that addition of pomegranate or its fraction as supplement would supply

little or no protein in the animal diet. The results pertaining to the effect of supplementation

of PoP, PM and PoPx on protein intake of experimental rats have been presented in Figures

4.7- 4.9.

The results of the present study demonstrated that the level of feed intake was

directly associated with the extent of protein intake in experimental rats fed pomegranate

peel and its fractions (Figure 4.7-4.9).Supplementing animal diet with PoPx at various

supplementation level did not present a significant change in protein intake in rats while

PoP and PM supplemented diet at 6 % supplementation level was shown to elevate protein

levels to 2.29g and 2.32g respectively in comparison with 2.53g increase in rats fed

unsupplemented diet (Figure 4.7 – 4.9). Type of supplements and the level of

supplementation, both seemed to contribute to show a variability in their effect to elevate

protein elevels in experimental rats. PoPx supplemented at a maximum 1.0%,being an extract

and not analogized with PoP and PM which were supplemeted at 6% apparently, lead to a

significant difference in protein intake level in rats. Current study further confirms

supplentation with plant material i.e. PoP and PM, leads to body weight reduction as

reported by Kim et al. (2012) who suggested plant proteins to be more effective in reducing

body weight owing to their ability to reduce volume of adipose tissues. The findings of the

present study open an avenue to further research on exploiting various fractions of

pomegranate peel in designing low protein formulas particularly for the treatment of

disorders like phenylketonuria.

110

Figure 4.7: Effect of PoP (2.0%), PM (2.0) and PoPx (0.25%) supplementation on protein intake



111

4.14.4 Effect of PoP, PM and PoPx supplementation on protein digestibility (%) in male

albino wistar rats

Data relating to the effect of PoP, PM and PoPx supplementation on protein

digestibility in rats have been presented in Figure 4.10 – 4.12.

The current study depicted a slight reduction in protein digestibility among rats fed of

PoP, PM and PoPx supplemented diets at various supplementation levels. Increased

supplementation level with PoP, PM and PoPx resulted in a decline in protein digestibility in

experimental rats. Tannins rich diets fed to laboratory animals are reportedly associated with

reduction in feed intake, product palatability and protein and carbohydrates digestibility

(Reed 1995).Maximum protein digestibility was recorded in rats supplied with PM (2.0, 4.0

and 6.0%) followed by those fed on PoPx (0.25, 0.50 and 1.0%) supplemented diets.

Slight declining trend was observed by increasing supplementation concentration of

PoPx among treated groups. The results depict a minor change in protein digestibility upto

3.1% among rats groups fed supplemented diets as compared to those fed control diets.

Pomegranate peel phenolics particularly the condensed tannins though constitute a very small

fraction of the fruit tannins, are likely to contribute in influencing the protein digestibility in

rats. Feeding ruminants with PoPx have been previously reported to halt protein and lipid

digestion non-significantly thereby affecting starch digestibility (Oliveira et al. 2010). Higher

protein digestibility in rats fed PoPx and PM supplemented diets may be associated with

lower levels of condensed tannins such as proanthocyanidins that are considered to be the

factor responsible for tannin - protein interaction.

112

Figure 4.12: Effect of PoP (6.0%), PM (6.0) and PoPx (1.00%) supplementation on protein digestibility



113

4.14.5 Effect of PoP, PM and PoPx supplementation on Feed Conversion Efficiency (FCE) in

male albino wistar rats

Feed conversion efficiency in treated vs control rats is presented in Figures 4.13 –

4.15. Present results revealed a gradual decline in feed conversion efficiency during feeding

period of 8 week. Feed conversion efficiency (FCE) calculated as weight gain /feed intake

per unit day recorded maximum FCE in rats fed 6.0% PM supplemented diet i.e. 0.112 –

0.102 as compared to those rats fed on standard diet (0.105 – 0.095). Rate of reduction in

FCE of rats fed on PoP gradually increased from 3.8 – 6.7% at 2 – 6% supplementation

whereas PoPx supplementation ~ 1.0% revealed no change in FCE at 1st to 8th week of study

period.

FCE as has been reported from previous findings is a function of higher protein and

caloric contents (Touchburn et al. 1981). PM, PoP, and PoPx on account of their least protein

contents and considerably low energy value were found to render poor FCE. The rats group

fed diet supplemented with PM (2.0 – 6.0%) also showed a 9.5% reduction in FCE at

maximum supplementation level. This study further concludes PM to possess better feed

conversion efficiency as compared to other tested groups during a sub-chronic exposure of 8

weeks.

One group of researchers reported an increase in FCE from -0.18 – 0.65 as a function

of increasing protein concentration from 0 – 30% (Mercer et al. 1981). Similarly, another

study elaborated how increased concentration of pomegranate peel (~7.5%) supplemented

pan bread fed to laboratory rats reduced feed intake, body weight and feed conversion

efficiency (Sayed – Ahmed 2014) which is in line with the results of the present study .

114

Figure 4.15: Effect of PoP (6.0%), PM (6.0) and PoPx (1.00%) supplementation on feed conversion efficiency

Figure 4.14: Effect of PoP (4.0%), PM (4.0) and PoPx (0.50%) supplementation onfeed conversion efficiency

Figure 4.14: Effect of PoP (2.0%), PM (2.0) and PoPx (0.25%) supplementation onfeed conversion efficiency

115

4.14.6 Effect of pomegranate peel meal supplementation on organ to body weight of

normal albino wistar rats

Organ weight might be a good indicator for toxicity evaluation of the feed ingredients

in rats model however; it has also been proposed that variation in organ weight may also be

associated to lower feed intake response of the experimental animals and hence must not be

confused with toxicity (Scharer, 1977). R except the heart, right lung and left kidney where

the data clearly depicted a significant effect of supplementation. Peak weight recorded for

heart at the end of study period was upto 10% lower than recorded in normal rats whereas a

gradual increment in weight was observed with progression of feeding duration.

Different treatments of PM were found to present non-significant effect of

supplementation on liver weight and maximum supplementation i.e. 6% was recorded to

increase the organ weight to 4.62g/100g body weight (b.w.) that was comparable to the one

recorded in control rats i.e. 4.622g/100g b.w. In comparison with the control, mild increment

in weight of right kidney (~5.0%) and left kidney (1.7%) was recorded at 6%

supplementation of PM. PM supplementation at various levels of supplementation did not

significantly affect spleen and lungs weight at maximum supplementation thereby

proclaiming the meal fraction of pomegranate peel as non-toxic to the experimental animal as

far as organ to body weight is concerned. As has been stated earlier in this section, significant

reduction in organ weight (heart, left kidney and right lung) might be associated to the

reduction in feed intake rather than the plant material mediated toxicity.

116

Table 4.22: Effect of pomegranate peel meal supplementation on organ to body weight (g) of

albino wistar rats at different time intervals

Organ Weight

(g/100g) Peel Meal

Study Intervals (days) Mean

0 28 56

Heart Control 0.489±0.0.005 0.510±0.002 0.532±0.006 0.510±0.015a

2.00% 0.420±0.023 0.427±0.018 0.459±0.019 0.435±0.15b

4.00% 0.429±0.009 0.441±0.017 0.469±0.015 0.446±0.014b

6.00% 0.428±0.007 0.453±0.009 0.478±0.022 0.453±0.018b

Mean 0.441±0.023c 0.458±0.026b 0.484±0.023a

Liver Control 4.292±0.055 4.590±0.052 4.622±0.043 4.501±0.129a

2.00% 4.197±0.050 4.384±0.151 4.555±0.160 4.379±0.127a

4.00% 4.156±0.070 4.437±0.161 4.509±0.179 4.367±0.132a

6.00% 4.288±0.094b 4.454±0.101a 4.618±0.084a 4.453±0.117a

Mean 4.233±0.048c 4.466±0.062b 4.576±0.038a

Spleen Control 0.242±0.004 0.264±0.006 0.282±0.009 0.262±0.014a

2.00% 0.240±0.014 0.251±0.013 0.263±0.009 0. 251±0.008a

4.00% 0.241±0.006 0.261±0.010 0.271±0.007 0.258±0.011a

6.00% 0.234±0.012 0.269±0.009 0.279±0.006 0.261±0.017a

Mean 0.239±0.003c 0.261±0.005b 0.274±0.006a

Right Kidney Control 0.341±0.006 0.371±0.009 0.397±0.006 0.376±0.020a

2.00% 0.344±0.012 0.361±0.016 0.401±0.015 0.372±0.021a

4.00% 0.346±0.013 0.371±0.011 0.410±0.021 0.378±0.023a

6.00% 0.344±0.009 0.381±0.017 0.417±0.018 0.387±0.026a

Mean 0.344±0.001c 0.371±0.006b 0.406±0.006a

Left Kidney Control 0.347±0.006 0.375±0.009 0.405±0.014 0.376±0.021ab

2.00% 0.353±0.013 0.373±0.011 0.389±0.010 0.372±0.013b

4.00% 0.346±0.013 0.383±0.011 0.405±0.012 0.378±0.021ab

6.00% 0.355±0.011 0.393±0.006 0.412±0.020 0.387±0.021a

Mean 0.350±0.003c 0.381±0.006b 0.403±0.007a

Right Lung Control 0.453±0.006 0.485±0.009 0.504±0.004 0.481±0.018a

2.00% 0.442±0.028 0.449±0.015 0.467±0.016 0.453±0.009b

4.00% 0.443±0.021 0.458±0.015 0.480±0.017 0.460±0.013b

6.00% 0.427±0.012 0.464±0.022 0.493±0.004 0.461±0.023ab

Mean 0.441±0.008c 0.464±0.011b 0.486±0.011a

Left Lung Control 0.225±0.009 0.247±0.006 0.285±0.009 0.252±0.021a

2.00% 0.235±0.020 0.257±0.019 0.266±0.013 0. 352±0.011a

4.00% 0.236±0.010 0.262±0.007 0.271±0.003 0.256±0.013a

6.00% 0.231±0.007 0.256±0.015 0.284±0.011 0.257±0.019a

Mean 0. 232±0.004c 0.255±0.004b 0.276±0.007a

Means sharing same letters in a row and column are not significantly different from each other at p<0.05.

117

4.14.7 Effect of pomegranate peel powder supplementation on organ to body weight of

albino wistar rats

Supplementation of pomegranate peel powder in rats’ diet and the feeding time of 28

days showed a significant effect on the weight of the heart however; level of

supplementation (2.0 – 6.0 %) did not indicate any significant (p< 0.05) change in this

organ’s weight (Table 4.23). PoP supplementation did not exert any change in the weight of

liver, spleen, right kidney and left kidney in contrary to the right and left lungs of the rats

which were shown to significantly reduce in weight irrespective of the level of

supplementation (Table 4.23). The data pertaining to the change in weight of these organs as

a function of supplementation demonstrated a significant effect of feeding supplemented

diet to the rats for a period of 56 days. In contrast to the other body organs except heart,

supplementation exhibited significant change in the weight of right and left lung of the rats

irrespective of supplementation level (Table 4.23)

In line with the findings from organ to body weight of albino wistar rats fed on meal

of pomegranate peel, highly significant effect of supplementation was noticed at different

time intervals (Table 4.23). Significant effect of PoP supplemented diet was identified for

heart and right lung of the rats whereas a non-significant effect was noticed for all other

organs.

Data given in Table 4.23 reveals stability of heart, liver and spleen weight after 28th

day of feeding whereas significant and gradual increment in the weight of both kidneys and

lungs was observed with increased feeding period i.e. upto 56days. Attributing to the organ

weight of control rats, PoP supplementation ~ 6% rendered reduction in heart weight (10%),

right kidney (~5%), left kidney (~3%), right lung (~3%) and left lung (~6%) that may be

associated with reduction in intake of PoP supplemented diet.

Moreover, PoP being low in protein contents are also expected to lower down protein

contents of the standard diet on supplementation that can further onset poor tendency of the

body to develop muscles and organ to body weight (de Castro and Boyd 1968). Physical

assessment of the organ studies identified lesser tendency of the animals to develop fatty

tissues around the organs proclaiming PoP tendency to mitigate fat deposition associated

obesity and reduction in organ weight. The study further suggested a competitive role of PoP

118

for not only maintaining body weight but also the organ weight of the studies animal as has

been observed for heart, liver and spleen.

Table 4.23: Effect of pomegranate peel powder supplementation on organ to body weight

(g) of wistar rats at different time intervals

Organ Weight

(g/100g) Peel Meal


0 28 56

Heart Control 0.489±0.005 0.510±0.002 0.532±0.006 0.510±0.015a

2.00% 0.433±0.012 0.455±0.012 0.462±0.014 0.450±0.011b

4.00% 0.426±0.014 0.463±0.012 0.469±0.015 0.453±0.016b

6.00% 0.430±0.018 0.473±0.015 0.477±0.009 0.460±0.018b

Mean 0.445±0.021b 0.475±0.017a 0.485±0.023a

Liver Control 4.292±0.055 4.590±0.052 4.622±0.043 4.501±0.129a

2.00% 4.127±0.093 4.435±0.215 4.616±0.204 4.393±0.175a

4.00% 4.237±0.165 4.502±0.115 4.661±0.109 4.467±0.151a

6.00% 4.239±0.119 4.543±0.125 4.615±0.198 4.466±0.141a

Mean 4.224±0.049b 4.518±0.046a 4.628±0.015a

Spleen Control 0.242±0.004 0.264±0.006 0.282±0.009 0.262±0.014a

2.00% 0.233±0.011 0.255±0.015 0.276±0.008 0. 255±0.015a

4.00% 0.227±0.067 0.260±0.005 0.279±0.015 0.255±0.019a

6.00% 0.234±0.011 0.265±0.011 0.278±0.006 0.259±0.016a

Mean 0.234±0.004b 0.261±0.003a 0.278±0.002a


2.00% 0.344±0.018 0.363±0.017 0.378±0.006 0.362±0.012a

4.00% 0.338±0.020 0.371±0.012 0.384±0.012 0.365±0.017a

6.00% 0.334±0.009 0.366±0.007 0.378±0.008 0.360±0.016a

Mean 0.339±0.003c 0.368±0.003b 0.384±0.006a

Left Kidney Control 0.347±0.006 0.375±0.009 0.405±0.014 0.376±0.021a

2.00% 0.350±0.006 0.367±0.012 0.379±0.009 0.365±0.010a

4.00% 0.351±0.009 0.375±0.014 0.382±0.010 0.369±0.011a

6.00% 0.350±0.028 0.381±0.022 0.394±0.011 0.375±0.016a

Mean 0.349±0.001c 0.375±0.004b 0.390±0.008a


2.00% 0.440±0.004 0.458±0.020 0.472±0.009 0.457±0.011b

4.00% 0.428±0.010 0.465±0.007 0.478±0.013 0.457±0.018b

6.00% 0.439±0.006 0.472±0.009 0.476±0.013 0.462±0.014b

Mean 0.440±0.007c 0.470±0.008b 0.483±0.010a

Left Lung Control 0.225±0.009 0.247±0.006 0.285±0.009 0.252±0.021a

2.00% 0.211±0.017 0.235±0.016 0.248±0.010 0. 232±0.013b

4.00% 0.227±0.016 0.245±0.010 0.258±0.008 0.244±0.011ab

6.00% 0.228±0.017 0.256±0.014 0.265±0.014 0.250±0.014a

Mean 0. 223±0.006c 0.246±0.006b 0.264±0.011a


119

4.14.8 Effect of pomegranate peel extracts supplementation on organ to body weight of

albino wistar rats

Data presented in Table 4.24 manifested a significant increase in the weight of heart,

left kidney, right lung and left lung of the rats fed on PoPx supplemented diet for 56 days

while such an increase in the weight of liver, spleen and right kidney did not continue after

28 days of feeding time. Similarly, supplementation itself had no influence on the weight of

liver, spleen, right kidney and right lung as no significant difference was noticed in the

weight of these organs whereas the weight of heart, left kidney and left lung was shown to be

significantly affected with changing supplementation level however; this change in organ

weight did not appear to take place in a dose dependent manner.

Feeding diet supplemented with different levels of PoPx to albino wistar rats for a

period of 56days offered a highly significant (p<0.01) effect of time interval on organ weight.

Significant effect of diet was noticed on the weight of heart (p<0.01) and left kidney (p<0.05)

whereas feeding rats with various levels of PoPx had produced non-significant effect on the

weight of liver, spleen, right kidney and, right and left lung. Gradual but non-significant

increase in organ weight was observed at 0.25, 0.50 and 1.0% supplementation that may be

referred as effect of adoptability to a newer diet and diet consumption.

Numerically, PoPx supplementation (1.0%) yielded upto 4% reduction in heart

weight as compared to the control after 56days of treatment. Highly non-significant effect of

treatments were observed on liver, right kidney, spleen and right as well as left lung

proclaiming PoPx to not capable of inducing any toxic effect at 1.0% supplementation on a

sub-chronic exposure for 56days. Reduction in heart weight may be associated with loss in

body weight (Oscai and Holloszy 1970) as has been observed in treated rats as compared to

the control. Reduction in heart weight has also been identified as function of body peak

weight gain. There are certain observation that heart weight index fall after body peak weight

gain.

120

Table 4.24: Effect of pomegranate peel extracts supplementation on organ to body weight

(g) of albino wistar rats at different time intervals

Organ Weight

(g/100g) Peel Meal


0 28 56

Heart Control 0.489±0.005 0.510±0.002 0.532±0.006 0.510±0.015a

0.25% 0.458±0.012 0.477±0.005 0.490±0.011 0.475±0.011c

0.50% 0.465±0.008 0.486±0.011 0.495±0.012 0.482±0.011bc

1.00% 0.461±0.015 0.496±0.008 0.512±0.007 0.490±0.018b

Mean 0.468±0.010c 0.492±0.010b 0.507±0.013a

Liver Control 4.292±0.055 4.590±0.052 4.622±0.043 4.501±0.129a

0.25% 4.127±0.108 4.513±0.034 4.627±0.077 4.469±0.185a

0.50% 4.332±0.179 4.493±0.064 4.572±0.069 4.465±0.086a

1.00% 4.287±0.100 4.462±0.085 4.537±0.055 4.429±0.091a

Mean 4.294±0.064b 4.515±0.039a 4.589±0.030a

Spleen Control 0.242±0.004 0.264±0.006 0.282±0.009 0.262±0.014a

0.25% 0.228±0.012 0.257±0.013 0.268±0.009 0. 251±0.015a

0.50% 0.240±0.020 0.264±0.010 0.272±0.013 0.259±0.012a

1.00% 0.233±0.012 0.269±0.006 0.279±0.007 0.260±0.017a

Mean 0.236±0.005b 0.264±0.003a 0.275±0.005a


0.25% 0.330±0.017 0.362±0.007 0.380±0.007 0.357±0.018a

0.50% 0.337±0.012 0.370±0.001 0.386±0.011 0.365±0.018a

1.00% 0.346±0.058 0.382±0.014 0.393±0.015 0.374±0.017a

Mean 0.339±0.005b 0.371±0.006a 0.389±0.005a

Left Kidney Control 0.347±0.006 0.375±0.009 0.405±0.014 0.376±0.021a

0.25% 0.327±0.016 0.362±0.010 0.381±0.006 0.356±0.019b

0.50% 0.335±0.018 0.372±0.009 0.387±0.008 0.365±0.019ab

1.00% 0.345±0.015 0.379±0.011 0.396±0.006 0.373±0.018a

Mean 0.338±0.007c 0.372±0.005b 0.392±0.007a


0.25% 0.455±0.034 0.471±0.017 0.491±0.009 0.472±0.013a

0.50% 0.448±0.013 0.480±0.007 0.493±0.010 0.474±0.016a

1.00% 0.457±0.016 0.485±0.010 0.496±0.011 0.479±0.014a

Mean 0.453±0.003c 0.480±0.005b 0.496±0.004a

Left Lung Control 0.225±0.009 0.247±0.006 0.285±0.009 0.252±0.021b

0.25% 0.241±0.007 0.261±0.015 0.275±0.012 0. 259±0.012ab

0.50% 0.230±0.012 0.255±0.014 0.278±0.010 0.254±0.017b

1.00% 0.246±0.009 0.270±0.007 0.292±0.011 0.269±0.016a

Mean 0. 235±0.007c 0.258±0.007b 0.282±0.005a


121

4.15 Serum Chemistry

4.15.1 Effect of feeding PoP, PoPx and PM on serum triglycerides levels of albino wistar rats

Higher serum triglycerides concentration is a moderate to highly significant risk

factor in development of coronary heart diseases (Sarwar et al. 2007). Toxicological

evaluation of pomegranate peel and its various fractions on serum lipid profile of albino

wistar rats indicated significant (p<0.05) effect of supplementation and the feed interval as

shown in ANOVA (Table 4.25).

A significant decline in serum triglycerides of the rats fed diets supplemented with

various levels of PoP, PM and PoPx was noticed. Data presented in Figures 4.16, 4.18

indicate that feeding diet supplemented with PoP and PoPx exert significant effect of

supplementation at 28th day of supplementation while no significant change in serum

triglycerides levels was noticed in the next 28 days of feeding. In comparison with the

control, maximum supplementation of PoP (6.00%) and PoPx (1.0%) revealed 5.1% and

5.9% reduction in serum triglycerides of albino rats. Conversely, PM supplementation for a

period of 56 days indicated ~1.6% increment in serum triglycerides i.e. 145.5 – 148.3mg/dL

(Figure 4.17).

In comparison with the control groups, the study exhibited a significant potential of

PoP and PoPx to reduce triglycerides level among the tested rats and this ability of PoP and

PoPx can be well exploited in hypercholesterolemia and associated disorders to keep lipid

profile at optimum levels . The study demonstrates PoP to hold relatively decreased tendency

in reducing serum triglycerides as compared to PoPx when supplied at 1.0% in rats’ diet.

Ellagic acid - a bioactive fraction of pomegranate and berries have been shown to reduce

triglycerides in normal rats by ~ 12.5% (106 – 92.7mg/dL) by oral supplementation of

1.25% bioactive fraction for a period of 90 days (Tasaki et al. 2008). The data obtained in the

present study indicated reduced potential of ellagic acid to reduce triglyceride levels as

compare to the results presented by Tasaki et al. (2008) and this difference might be

attributed to crude form of extracts and the peel powder used in present research.

122

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00

0 Days 28 Days 56 DaysTri

gly

ceri

des (

mg

/dL

)

Control 2% Peel Powder 4% Peel Powder 6% Peel Powder

Level of significance (Treatments: <0.05ns, Intervals: <0.05ns)

Figure 4. 16: Effect of PoP supplementation on serum triglycerides (mg/dL) levels of albino

wistar male rats

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00


gly

ceri

desl

(mg

/dL

)

Control 2% Peel Meal 4% Peel Meal 6% Peel Meal

Level of significance (Treatments: >0.05ns, Intervals: >0.05ns)

Figure 4. 17: Effect of PM supplementation on serum triglycerides (mg/dL) levels of albino

wistar male rats

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00


gly

ceri

des

l (m

g/d

L)

Control 0.25% Peel Extract 0.50% Peel Extract 1.0% Peel Extract

Level of significance (Treatments: <0.05ns, Intervals: <0.05ns)

Figure 4. 18: Effect of PoPx supplementation on serum triglycerides (mg/dL) levels of albino

wistar male rats

123

4.15.2 Effect of feeding PoP,PoPx and PM on serum total cholesterol levels of albino wistar

rats

Hypercholesterolemia is a major modifiable risk factor for development of coronary

heart diseases. Data are available to suggest coronary heart diseases to be associated with

high rate of morbidity and mortality, leading to huge public health and economic losses

worldwide (Pearson et al. 2002; Ford et al. 2003). Pomegranate and its various anatomical

segments have been found to possess potential lipid modulatory features in the last few

decades (Neyrinck et al. 2013).

The results obtained from the present research portray PoPx supplementation to have

significantly affected the total cholesterol levels among rats as a function of feeding interval

and treatment combination (Table 4.25). Contrarily, the group of experimental rats fed on

PM did not show any significant change in serum cholesterol level with respect to

supplementation levels and feeding interval while the rats supplied PoP supplemented diet

(2.0 – 6.0%) showed significant effect of supplementation in their serum cholesterol level

(Figure 4.19-4.21). Feeding PoPx supplemented diet to rats for a period of 56 days indicated

~1.7% reduction in total cholesterol contents among various groups (Figure 4.21).

Comparison of various feed groups for evaluating efficiency of diet and supplementation

levels on serum cholesterol in rats indicated significant effect of PoP at 6.0%

supplementation while PoPx above 0.5% supplementation non-significantly affected

cholesterol contents of the rats.

PM supplemented diet, being rich in fiber content, did not seem to show a significant

effect on curtailing serum cholesterol level among tested rats as compared to those supplied

with diet rich in pomegranate peel phenolics. This may be associated with higher

concentration of non-hydrolysable fibers in PM and their poor tendency to bind and eliminate

cholesterol from the animal gut.

124

Approximately 5% reduction in total cholesterol contents have been previously

reported among rats fed on 6% punicalagin diet for a period of 37days (Cerda et al. 2003b).

The concentration of punicalagin dispensed to the rats as reported by Cerda et al (2003b)

seems to be much higher than that of bioactive fractions of PoP, PoPx and PM suggesting

relatively lower rate of decline in triglycerides and total cholesterol levels among the tested

rats.

125

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00

0 Days 28 Days 56 DaysTo

tal C

ho

leste

rol

(mg

/dL

)


Level of significance (Treatments: <0.05, Intervals: >0.05ns)

Figure 4. 19: Effect of PoP supplementation on serum total cholesterol (mg/dL) levels of albino

wistar male rats

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00

0 Days 28 Days 56 DaysTo

tal C

ho

leste

roll

(m

g/d

L)



Figure 4. 20: Effect of PM supplementation on serum total cholesterol (mg/dL) levels of albino

wistar male rats

120.00

125.00

130.00

135.00

140.00

145.00

150.00

155.00

0 Days 28 Days 56 Days

To

tal C

ho

leste

rol

(mg

/dL

)


Level of significance (Treatments: <0.05, Intervals: <0.05)

Figure 4. 21: Effect of PoPx supplementation on serum total cholesterol (mg/dL) levels of albino

wistar male rats

126

4.15.3 Effect of feeding PoP,PoPx and PM on serum HDL levels of albino wistar rats

High density lipoprotein (HDL) ordinarily constitutes around 20% of the plasma

cholesterol. Evidence based studies highlight significant association of lower serum HDL

concentration and high risks of coronary heart diseases in healthy and ischemic individuals.

Anti-atherogenic properties of HDL are coupled with their active participation in cholesterol

transport reversal, minimizing risks of endothelial dysfunction and preventing oxidative

stress (Assmann and Nofer 2003). Nutraceuticals such as those derived from pomegranate

have been shown to bear promising characteristics to protect and improve serum HDL

reserves in normal and hypercholesterolemic subjects (Asgary et al. 2013; Houston 2012).

Supplementing PM and PoPx in rats’ diet manifested a significant effect of

supplementation on serum HDL concentration however PoP supplemented diet did not

exhibit such effect among the tested rats. Our results showed no effect of feeding time of

various supplemented diets to rats HDL levels (Table 4.25). The results further corroborated

that feeding diet supplemented with 1.0% PoPx improved serum HDL concentration of the

rats by 2.3% i.e. 40.48 - 41.43 mg/dL while relatively lower and non-significant increment

i.e. ~1.7% and 1.3% was noticed for PoP and PM supplementation, respectively. Feeding rats

with either PoP, PM or PoPx supplemented diets at 28 to 56 days period did not significantly

affect HDL levels of the albino wistar rats (Figure 4.22-4.24).

Evaluation of the toxicity of pomegranate peel has been a focus of this part of the

study. Our findings depicted no toxic response in terms of an abnormal change in serum

HDL contents of normal rats. One retrospective study demonstrated a significant increment

in HDL concentration (~9.0%) in dislipidemic human subjects on ingestion of seed oil of

pomegranate (Asghari et al. 2012). Another study from Isfahan, Iran delineated

hypolipidemic role of pomegranate juice in hypertensive patients by increasing HDL

concentration ~1.1% (Asgary et al. 2013). PoP, being evaluated for its anti-atherogenic

activity in rabbits have also been reported to improve serum HDL concentration by 21% that

is too higher than that of, reported in this study.

127

25.00

29.00

33.00

37.00

41.00

45.00


HD

L (

mg

/dL

)


Level of significance (Treatments: >0.05ns, Intervals: Level of significance (Treatments: >0.05ns, Intervals:

Figure 4. 22: Effect of PoP supplementation on High Density Lipoprotein (mg/dL) levels of

albino wistar male rats

25.00

29.00

33.00

37.00

41.00

45.00


HD

L (

mg

/dL

)


Level of significance (Treatments: >0.05ns, Intervals:

Figure 4. 23: Effect of PM supplementation on High Density Lipoprotein (mg/dL) levels of


25.00

29.00

33.00

37.00

41.00

45.00


HD

Ll

(mg

/dL

)


Level of significance (Treatments: <0.05, Intervals:

Figure 4. 24: Effect of PoPx supplementation on High Density Lipoprotein (mg/dL) levels of


128

4.15.4 Effect of feeding PoP,PoPx and PM on serum LDL levels of Albino wistar rats

LDL is a predominant marker in cardiac health where elevated levels of the

compound stand responsible for multiple cardiovascular diseases. The ability of PoP and its

extracts to reduce LDL levels in hypercholesterolemic rats has been reported with their slight

but significant tendency to reduce LDL contents among these rats. Analysis of variance for

serum LDL contents of rats fed on PoP, PM and PoPx supplemented diet presents significant

effect of supplementation (Table 4.25) however; feeding interval did not exhibit any

significant effect on serum LDL concentration of rats for PM and PoPx supplemented diets

exception being PoP supplemented diet. Data presented in Figure 4.25 -4.27 indicate a

decline in LDL concentration of rats fed on varying levels of PoP, PM and PoPx in their

diets. Highest rate of reduction in LDL concentration of rats was noticed in PoPx group

where 1.0% supplementation resulted in 3.3% reduction in LDL as compared to the control

followed by PoP where rate of LDL reduction was noticed to be 2.57%. Rats fed on PM

supplemented diet exhibited significant reduction in their LDL contents at 2.0%

supplementation whereas non-significant decline was observed at 4.0 and 6.0%

supplementation levels.

Effect of PoPx has been previously evaluated in high lipid diet fed rats signifying a

considerable reduction in serum LDL concentration. One study reported PoPx

supplementation at 500mg/kg b.w. for 23days to lower down the LDL levels of

hypercholesterolemic rats as compared to the normal rats i.e. 92 to 61 mg/dL (Sadeghipour et

al. 2014). In line with the findings of current study, a double-blind controlled trial on

administration of pomegranate juice to dialysis patients for one year demonstrated a

reduction in serum LDL concentration by 1.2% as compared to the placebo (Shema-Didi et

al. 2012). A visible role of pomegranate juice in reducing LDL has also been reported by

Anoosh et al. (2012) revealing 4 week treatment of hypercholesterolemic patients with the

fruit juice to lower down serum LDL contents by 17.5%. LDL ability of pomegranate, its

juice, peel and seed fractions to reduce LDL contents, are attributed to their unique phenolic

composition comprising a wide range of high molecular weight biomolecules.

129

40.00

50.00

60.00

70.00

80.00


LD

L (

mg

/dL

)



Figure 4. 25: Effect of PoP supplementation on Low Density Lipoprotein (mg/dL) levels of


40.00

50.00

60.00

70.00

80.00


LD

L (

mg

/dL

)



Figure 4. 26: Effect of PM supplementation on Low Density Lipoprotein (mg/dL) levels of


40.00

50.00

60.00

70.00

80.00


LD

Ll (m

g/d

L)


Level of significance (Treatments: <0.05, Intervals: >0.05ns)

Figure 4. 27: Effect of PoPx supplementation on Low Density Lipoprotein (mg/dL) levels of


130

4.15.5 Effect of PoP, PM and PoPx supplementation on kidney and liver function properties

of albino wistar rats

4.15.5.1 Uric Acid

Sufficient literature is available to establish chronic elevated levels of serum uric

acid to be substantially associated with gout, renal calculi, metabolic syndrome and cardiac

disorders (de Olivera and Brurini 2012). Accumulation of uric acid in blood is an indicator

of its impaired secretion or elevated synthesis. The results of the present study validated a

mediating role of PoP and its fractions in hyperuricemia. The data presented in Table 4.25

showed PoP and PoPx supplemented diets to produce significant effect with respect to the

treatments and study intervals (0 – 56 days) on serum uric acid levels in normal rats.

Our findings indicate a comparative effect of PoP and PoPx supplementation in

lowering serum uric acid concentration of treated rats as compared to the PM supplemented

group (figure 4.28 – 4.30). Maximum decline in serum uric acid level was observed in PoP

and PoPx supplemented group where 56 days feeding period resulted in a decline in uric

acid levels from 2.36 – 2.31mg/dL whereas, uric acid level among PM supplemented groups

was reduced from 2.37 – 2.35mg/dL (Figure 4.29)

Results pertaining to the ability of PoPx and PoP supplemented diets at 1.0% and

6.0% supplementation levels indicated variability in their ability to reduce serum uric acid

levels among experimental animals. Around 1.5% reduction in uric acid concentration was

noticed in PM supplemented group at highest level of PM supplementation (7.5%). Non-

significant change was observed in uric acid concentration of rats fed PoP and PM

supplemented diets after 28 days of feeding period. Vidal et al. (2003) explicated the impact

of administering pomegranate extracts on serum uric acid levels in rats thereby suggesting

upto 26.6% reduction in serum uric acid level as compared to control on intraperitoneal

administration of 25 – 2000mg of pomegranate extracts (including peel fraction) to normal

rats. Current research demonstrated relatively lower reduction rates of uric acid probably due

to oral feeding of PoP, PM and PoPx of Alipuri cultivar.

131

1.50

1.70

1.90

2.10

2.30

2.50


Uri

c a

cid

(m

g/d

L)



Figure 4. 28: Effect of PoP supplementation on Uric Acid (mg/dL) levels of albino wistar male

rats

1.50

1.70

1.90

2.10

2.30

2.50


Uri

c a

cid

(m

g/d

L)



Figure 4. 29: Effect of PM supplementation on Uric Acid (mg/dL) levels of albino wistar male

rats

1.50

1.70

1.90

2.10

2.30

2.50


Uri

c a

cid

(m

g/d

L)



Figure 4. 30: Effect of PoPx supplementation on Uric Acid (mg/dL) levels of albino wistar male

rats

132

4.15.5.2 Serum Creatinine

Creatinine is a chemical waste produced by the body in healthy muscle metabolism.

Creatinine removal from the body is a normal kidney function process however; elevated

serum creatinine levels generally indicate malfunctioned glomerular filtration and impaired

kidneys. Pomegranate, the fruit juice, peel extracts and the seed oil have properties to reduce

renal toxicity vide inhibition of glutathione, nitric oxide and malondialdehyde production

ultimately imparting efficient waste clearing properties to the kidneys (Tugcu et al. 2008; Al-

Gubory et al. 2015).

Present research indicated a tendency in PoP and PoPx to improve serum creatinine

levels of albino wistar rats. Analysis of variance (Table 4.25) showed a highly significant

response of study period i.e. feeding time and supplementation levels on serum creatinine

levels of PoP and PoPx supplemented diet groups. A consistent decline in creatinine contents

of rats fed PoP supplemented diet was observed from 0 – 56 days of study whilst creatinine

contents did not significantly change amongst the rats fed on PM supplemented diet after 28th

day of study. The groups and treatments comparison revealed PoPx to exhibit maximum

reduction trend for creatinine i.e. 12.3% followed by 8.9% from rats provided with PoP

supplemented diet (Figure 4.31- 4.33). Supplementation of PoPx in the diet has shown to

affect the creatinine levels as shown in figure 4.33. The comparative results i.e. creatinine

levels of 0.50mg/dL for normal rats and 0.44mg/dL for rats supplied PoPx supplemented

diets, indicate that supplementation of PoPx holds the ability to reduce creatinine level in rats

(Figure 4.33). Variability in serum creatinine levels among tested rats, as a function of

supplementation with 2 – 6% PM remained to be the minimum. The meal fraction, being a

carrier of very lower residual levels of pomegranate phenolics when offered for consumption

as rats’ feed, exhibited relatively lower tendency to reduce serum creatinine level as

compared to the other two fractions i.e. PoP and PoPx.

Rate of decline in rats’s serum creatinine level at maximum supplementation level of

PoP, PM and PoPx as observed in current study, are lower to that of reported by Cerda et al.

(2003a) who studied safety assessment of punicalgin – a bioactive fraction of pomegranate

and reported punicalgin to lower down serum creatinine contents from 0.5mg/dL (control) to

0.40mg/dL (punicalagin treated) after 37days feeding of 6% punicalagin supplemented feed.

133

Our results however showed a decreased reduction in creatinine levels as a result of feeding

PoP, PM and PoPx through diet and this difference might be attributed to PoP, PM and PoPx

being biologically less active crude forms of pomegranate fractions.

0.10

0.20

0.30

0.40

0.50

0.60


Cre

ati

nin

e (

mg

/dL

)



Figure 4. 31: Effect of PoP supplementation on creatinine (mg/dL) levels of albino wistar male

rats

0.10

0.20

0.30

0.40

0.50

0.60


Cre

ati

nin

e (

mg

/dL

)



0.10

0.20

0.30

0.40

0.50

0.60


Cre

ati

nin

e (

mg

/dL

)



Figure 4. 32: Effect of PM supplementation on creatinine (mg/dL) levels of albino wistar male

rats

Figure 4. 33: Effect of PoPx supplementation on creatinine (mg/dL) levels of albino wistar male

rats

134

4.15.5.3 Serum bilirubin contents

Bilirubin, generally referred as a heme moiety that originates from heme containing

protein and might exists in conjugated or un-conjugated forms. Increased levels of bilirubin

or lower excretion of the waste fraction are reportedly associated with increased hemolysis,

erythropoiesis, obstruction in excretion and impaired metabolism (Lester and Schmid 1964).

Pomegranate and its various fractions particularly the peel segments are well known

ingredient of ethnopharmacological preparations to mitigate certain chronic ailments

including those of hepatic carcinomas. Our study on pomegranate peel and its fraction

primarily focused upon assessing the impact of such ingredients on the safety of the foods

these ingredients might be used in. The results obtained to find out the toxicological limits of

these ingredients demonstrated that, supplementing PoP and its other fractions to albino

wistar rats’s diet for an extended period of 56 days depicted a highly significant effect on

serum bilirubin contents.

Feeding rats with PoPx and PoP supplemented diet significantly reduced serum

bilirubin contents consistently between 0 – 56 days while PM supplementation indicated non-

significant decline in bilirubin contents at 56th day of feeding such diet (Table 4.25). Rate of

bilirubin levels reduction was highest among PoPx treated groups followed by PoP and PM

i.e. 4.59%, 3.53% and 1.43%, respectively. Treatments comparison within the group revealed

PoPx supplementation at 1.0% level to reduce bilirubin levels by 4.82%. Increasing PoPx

concentration in animal diet above 0.25% did not show significant change as compared to the

control (Figure 4.34 - 4.36). Our findings further substantiated the ability of PoP to decrease

bilirubin contents from 0.77 – 0.74mg/dL at 6.0% supplementation in animal model whereas

significant but a moderate decline i.e. 0.77 – 0.76mg/dL in bilirubin contents was noticed at 0

– 6% supplementation level of PM.

135

Safety assessment of pomegranate extracts reported by by Cerda et al. (2003b)

suggested oral supplementation of 600 mg of pomegranate fruit extracts to lower down

bilirubin contents from 0.16 – 0.10 mg/dL after a sub-chronic exposure for 90 days. In

agreement with the findings of Cerda et al. (2003b), PoP and its extracts supplementation

reduced bilirubin levels in normal rats but the levels reached were quite in range for the

normal rats.

136

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80


Bilir

ub

in (

mg

/dL

)



Figure 4. 34: Effect of PoP supplementation on bilirubin (mg/dL) levels of albino wistar male

rats

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80


Bilir

ub

inl

(mg

/dL

)



Figure 4. 35: Effect of PM supplementation on bilirubin (mg/dL) levels of albino wistar male

rats

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80


Bilir

ub

in (

mg

/dL

)



Figure 4. 36: Effect of PoPx supplementation on bilirubin (mg/dL) levels of albino wistar male

rats

137

4.15.5.4 Serum Alanine Aminotransferase

Alanine aminotransferase (ALT) is an enzyme comprising of 496 amino acid and is

responsible for catalyzing amino groups transfer to produce oxaloacetates. ALT are

abundantly located in hepatocytes cytosol and hence their activity is 3000times higher in

liver than in serum. Hepatocellular injury or damage releases ALT into the serum and hence

it is recognized as a very important marker to identify hepatic injury (Kim et al. 2008).

The data analysis for serum chemistry of PoP, PM and PoPx supplemented diet fed

rats reveals dietary supplementation of pomegranate fractions to offer non-significant effect

on rat’s serum alkaline transaminase (ALT) contents (Table 4.25). Non-significant effect of

feeding interval and supplementation levels interaction was observed on serum ALT of rats

in all study groups. Contrarily, PoP and PM supplemented diet fed rats, the study presented a

significant (p<0.05) effect of PoPx supplementation levels on rat’s ALT levels.

Comparative levels of the ALT for control rats and those fed varying levels of PoP,

PM and PoPx are presented in Figures 4.37 - 4.39. The study presented a non-significant

(p>0.05) effect of feeding interval i.e. 0, 28 and 56 days on serum ALT contents of rats fed

PoP, PM and PoPx supplemented diet. PoP, PM and PoPx supplementation in rats diets

revealed 0.71, 0.17 and 0.88% (p>0.05) reduction in ALT, respectively during 56days

feeding period. The treatments comparison for various groups further revealed PoPx

supplementation @ 1.0% to offer maximum decline in ALT contents i.e. from 65.9 – 64.73

IU/L at 56th day of feeding.

Overall, a slight decline in serum ALT concentration of normal rats at 0 – 56days of

feeding with PoP, PM and PoPx supplemented diet and their various level indicates least to

no toxicity of the plant material on serum enzymes activities as has been evidently identified

in current report.

138

In comparison with the findings of current study, a far higher ALT reduction trend

has been recorded by Patel et al. (2008) from the pomegranate fruit extracts without any

evident sign or symptom of toxicity on the feeding animal. This group of researchers

interprets increasing supplementation quantity of pomegranate extracts i.e. from 0 –

600mg/kg to be responsible for a gradual decline in serum ALT concentration (71.5 –

47.39IU/L). The study further delineates serum ALT lower levels among the treated rats yet

to fall in safe range for the biomarker.

139

20.00

30.00

40.00

50.00

60.00

70.00

80.00


AL

T (

IU/L

)



20.00

30.00

40.00

50.00

60.00

70.00

80.00


AL

T (

IU/L

)



20.00

30.00

40.00

50.00

60.00

70.00

80.00


AL

Tl

(IU

/L)



Figure 4. 38: Effect of PM supplementation on ALT (mg/dL) levels of albino wistar male rats

Figure 4. 37: Effect of PoP supplementation on ALT (mg/dL) levels of albino wistar male rats

Figure 4. 39: Effect of PoPx supplementation on ALT (mg/dL) levels of albino wistar male rats

140

4.15.5.5 Serum Aspartate Aminotransferase

Formerly known as serum glutamic oxaloacetic transaminase(SGOT), Serum

Aspartate Aminotransferase (AST) is predominately located in liver, skeletal muscles, heart

and kidney. Increasingly higher levels of AST are reported due to liver damage, myocardial

infarction, hepatitis and chemical poisoning where the enzymes are released from the

damaged cell to the serum and subsequently builds serum AST levels.

Analysis of variance of data generated on serum AST contents of albino wistar rats

feed on various levels of PoP, PM and PoPx indicated non-significant (p>0.05) effect of

feeding interval on rat’s serum AST concentration. Seemingly, varying levels of PoP and PM

also presented a non-significant effect on serum AST whereas rats fed PoPx supplemented

diet at a concentration ranging between 0.25 – 1.0% significantly affected AST levels (Table

4.25).

In line with the findings on serum ALT concentrations, rats fed on diet carrying PoP,

PM and PoPx for a period of 0 – 56days were identified with a non-significant decline in

AST contents as compared to the control (Figure 4.40 and 4.41). Mean comparison of AST

levels in PoP, PM and PoPx fed rats groups revealed maximum decline (i.e. ~1.0%) in serum

AST concentration in PoPx fed rats further showing PoP to possess relatively higher

tendency to reduce AST concentration i.e. ~0.7% than PM (0.34%) at 56days of sub-chronic

feeding. Treatments comparison among the groups (PoP, PM and PoPx) indicated significant

reduction (~1.54%) in serum AST contents i.e. from 74.44 – 73.30IU/l of rats fed 1.0% PoPx

supplemented diet.

As observed for serum ALT level in rats model, Patel et al. (2008) explicated a

gradual declining trend (138.8 – 116.1 IU/l) on serum AST levels of male rats at 0 –

600mg/kg pomegranate fruit extracts supplementation. A reduction in AST levels of the rats

feed on various by-fractions of pomegranate peel for a sub-chronic period of 56days

represents a least toxic nature of the plant material on its judicious application in various

consumable goods at the rate of ~1.0% for PoPx and ~6.0% for both the PoP and PM.

141

20.00

30.00

40.00

50.00

60.00

70.00

80.00


AS

T (

IU/L

)



Figure 4. 40: Effect of PoP supplementation on AST (mg/dL) levels of albino wistar male rats

20.00

30.00

40.00

50.00

60.00

70.00

80.00


AS

T (

IU/L

)



Figure 4. 41: Effect of PM supplementation on AST (mg/dL) levels of albino wistar male rats

20.00

30.00

40.00

50.00

60.00

70.00

80.00


AS

T (

IU/L

)



Figure 4. 42: Effect of PoPx supplementation on AST (mg/dL) levels of albino wistar male rats

142

4.15.5.6 Serum alkaline phosphatase

Alkaline phosphatase (ALP) is predominately of hepatic origin and is also known to

be present in bone, kidney and placental tissues. Abnormally higher levels of ALP in serum

indicate hepatic disorder linked with obstruction of bile outflow and bone diseases having

higher osteoblast activity (McMaster et al. 1962). Normal range of ALP for controlled male

is 56 – 216IU/L with a mean value of 137IU/L (Charles River Laboratories 1998).

Pomegranate, the fruit and its extracts have been well admired for their hepatoprotective role

and maintaining liver enzymes balance (Faria et al. 2007).

Table 4.25 indicates non-significant effect of feeding intervals on serum ALP

concentration of rats fed various concentration of PoP, PM and PoPx. However, PoP, PM and

PoPx supplementation in rats diet exhibited a significant effect of treatment levels on serum

ALP. Mean comparison of ALP levels among various rats groups revealed non-significant

decline in ALP when exposed to PoP, PM and PoPx supplemented diet for a period of

56days (Figure 4.43 – 4.45). Maximum reduction in serum ALP concentration was noticed in

PoP fed rats where rate of reduction was 0.53% at 56 days of feeding (p>0.05) while PoPx

and PM group presented 0.5 and 0.3% decline in ALP, respectively as compared to those

tested at 0day. The data presented in Table 4.34 further indicate lower levels of PoP (2.0%),

PM (2.0%) and PoPx (0.25%) supplementation to exert a significant effect of feeding

supplemented diet as compared to the control whereas non-significant effect of above 2.0%

(PoP, PM) and 0.25% (PoPx) dietary supplementation was noticed in rats’ serum thereby

suggesting a decrease potential to significantly alter enzymes levels in normal rats. Hepatic

enzymes protection from oxidative stress is attributed to pomegranate phenolics particularly

punicalagin, ellagic acid and flavonoids. In a safety assessment study on normal rats, ellagic

acid has been reported to reduce ALP levels by 8% at 1.25% supplementation (Tasaki et al.

2008). The findings of the present study signify relatively lower potential of pomegranate

peel and its extracts in altering ALP levels of normal rats.

143

Figure 4. 43: Effect of PoP supplementation on ALP (mg/dL) levels of albino wistar male rats

80.00

100.00

120.00

140.00

160.00


AL

P (

IU/L

)



80.00

100.00

120.00

140.00

160.00

180.00


AL

P (

IU/L

)



Figure 4. 44: Effect of PM supplementation on ALP (mg/dL) levels of albino wistar male rats

80.00

100.00

120.00

140.00

160.00

180.00


AL

P (

IU/L

)



Figure 4. 45: Effect of PoPx supplementation on ALP (mg/dL) levels of albino wistar male rats

144

4.15.6 Serum total protein contents

Serum total protein is a measure of all fractions of protein including those of

immunoglobulin and it is intermittently considered a marker for liver function test where the

higher serum total protein levels indicate abnormal generation of immunoglobulin. Reference

range for serum total protein in 7 – 8 week rats has been reported in a range of 5.4 –

6.5mg/dL (Zaias et al. 2009). Safety assessment study of PoP, PM and PoPx for a period of

56 days revealed total protein contents of the experimental rats model to fall in prescribed

range.

Trends on serum total protein contents of rats fed PoP, PM and PoPx supplemented

diets are presented in Figures 4.46 – 4.48. Analysis of variance (Table 4.25) presents non-

significant effect of PoP, PM and PoPx supplementation during a feeding period of 56 days.

Rats supplied diets supplemented with various fractions of pomegranate peel presented non-

significant effect of feeding interval and interaction between treatment and feeding interval

on serum total protein contents (Figure 4.46 – 4.48).

The results of the present study indicated ~0.42% increment in total protein contents

of rats fed 6% PM supplemented diet. Likewise, the rate of increment in total protein

contents of rats supplied PM supplemented diet during a period of 56days feeding was

observed to be 0.2%. PoPx supplemented diet did not exert any significant effect on total

protein contents in rats though a slight decline in total protein contents of rats i.e. from 5.45

– 5.44mg/dL was noticed at maximum supplementation level i.e. 1.0%.

Slight increase in serum total protein content in rats as depicted in the present study

might be due to inadequate water consumption as needed by the body or mildly higher water

loss could be another reason for total protein increment. Biologically safe but comparatively

higher serum total protein contents in male rats (7.18g/dL) fed on 600g/kg pomegranate fruit

extracts comparing with control rats (7.07g/dL) were reported by Patel et al. (2008). The

present results are a proxy of the safety status of PoP, PoPx and PM from Alipuri white rind

cultivars. .

145

2.00

3.00

4.00

5.00

6.00

7.00


To

tal P

rote

in (

g/d

L)



Figure 4. 46: Effect of PoP supplementation on total protein (mg/dL) levels of albino wistar

male rats

2.00

3.00

4.00

5.00

6.00


To

tal P

rote

in (

g/d

L)



Figure 4. 47: Effect of PM supplementation on total protein (mg/dL) levels of albino wistar male

rats

2.00

3.00

4.00

5.00

6.00


To

tal P

rote

inl

(g/d

L)



Figure 4. 48: Effect of PoPx supplementation on total protein (mg/dL) levels of albino wistar

male rats

146

4.15.7 Serum albumin contents

Serum albumin, a single peptide of around 580 residues, is a plentiful plasma protein

responsible for multiple physiological properties including balancing blood osmotic pressure,

transportation of fatty acids and, bilirubin sequestering and its transportation (Peters, 1995).

Our findings on serum albumin contents among rats supplied diets supplemented

with various fractions of PoP, PM and PoPx were found to show identical trends for serum

total protein contents (Figure 4.49 – 4.51). Feeding PoP, PM and PoPx to albino wistar rats

indicated non-significant effect of supplementation at 2 – 6% of PoP and PM and, 0.25 –

1.0% supplementation level of PoPx in all groups of rats whereas non-significant effect of

feeding interval and interaction between feeding interval and supplementation level was

noticed in the present study (Table 4.25 ).

Feeding rats with PoP supplemented diet presented a non - significant decline in

serum albumin at 6.0% supplementation level. Similar trend was noticed in PM and PoPx

group where non-significant change in serum albumin contents of rats was observed at

lowest to highest supplementation level i.e. 2.0 – 6.0% (PM) and 0.25 – 1.0% (PoPx) (Figure

4.50 – 4.51). Comparative assessment of feeding pomegranate peel fractions revealed PoP to

present maximum rate of reduction in serum albumin contents of rats i.e. 0.66% followed by

PoPx producing 0.24% reduction at 1.0% dietary supplementation.

Serum albumin concentration in rats fed on PoP and PoPx portrays identical trends

with those reported by Patel et al. (2008). These researchers supplied pomegranate fruit

extracts as supplement at 60 mg/kg to rats and noticed serum albumin contents higher than

those observed in normal rats whereas higher dose of supplementation (600mg/kg) for the

same course of time showed a slight reduction (~0.3%) in serum albumin as compared to the

control. The study suggested PoP and PoPx supplementation in rats diet resulted in a slight

reduction trend in serum albumin and total protein contents in comparison with the control

whereas PM supplementation in rats diet induced a mild addition in serum protein value of

experimental animals without showing any physical symptom of toxicity.

147

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00


Alb

um

inl

(g/d

L)



Figure 4. 49: Effect of PoP supplementation on total albumin (mg/dL) levels of albino wistar

male rats

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00


AL

bu

min

(g

/dL

)



Figure 4. 50: Effect of PM supplementation on total albumin (mg/dL) levels of albino wistar

male rats

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00


Alb

um

in

(g/d

L)



Figure 4. 51: Effect of PoPx supplementation on total albumin (mg/dL) levels of albino wistar

male rats

148

4.15.8 Serum Glucose

Glycemic response of inedible plant material incorporated in food formulations as

dietary ingredient is also an important segment of safety assessment study. Impaired

secretion of insulin or otherwise its in-sensitization induces accumulation of higher levels of

glucose in blood. Plant extracts, owing to their residual toxic compounds may stand

responsible for pancreatic toxicity in the form of beta cells dysfunction and insulin resistance.

Moreover, chronic liver diseases – a hepatotoxicity response, have also been shown to

reportedly linked with glucose intolerance (Megyesiet al. 1967).

The fruit and peel extracts of pomegranate are reportedly associated with

management of Type -2 diabetes and the key mechanism behind diabetes protective role of

the pomegranate phenolics is inhibition of lipid peroxidation and oxidative stress. Beside its

pancreatic beta cells protective features, upper levels of PoP (6.0%), PoPx (1.0%) and PM

(6.0%) incorporated in ready to eat food products, were tested for any of their toxic effect on

glycemic response of rats model.

Data presented in Table 4.25 indicate PoP, PoPx and PM supplemented in rats’ diet at

various levels and fed for a period of 56 days did not significantly affect serum glucose

levels. Comparison of means for 0, 28 ad 56 days feeding intervals revealed non-significant

effect of feeding time with each other (Figure 4.52 – 4.54). Similar trend was noticed while

comparing groupsand the supplementation levels in each group where PoP, PoPx and PM did

not exert any significant change in serum glucose levels in comparison with the control. The

study revealed that maximum supplementation levels of PoP (6.0%) and PoPx (1.0%) to non-

significantly affect serum glucose with least difference from that of the control thereby

declaring both hyper nor hypoglycemic response of PoP and PoPx supplementation in normal

rats. Pomegranate meal (PM) was shown to slightly increase serum glucose concentration i.e.

~0.24% of the normal rats at 6.0% supplementation as compared to the control. Non-

significant response of feeding pomegranate extracts to the normal rats has been previously

reported by Patel et al. (2008). The findings of current study are in line with those of Patel et

al. (2008) that refer feeding pomegranate fruit extracts ~600mg/kg per day to the male rats

for a period of 90days to not induce any abnormal increase or decrease in serum glucose

levels.

149

80.00

90.00

100.00

110.00

120.00

130.00


Glu

co

se (

mg

/dL

)



80.00

90.00

100.00

110.00

120.00

130.00


Glu

co

sel

(mg

/dL

)


Level of significance (Treatments: <0.05ns, Intervals: >0.05ns)Level of significance (Treatments: >0.05ns, Intervals: >0.05ns)

Figure 4. 52: Effect of PoP supplementation on Glucose (mg/dL) levels of albino wistar male

rats

Figure 4. 53: Effect of PM supplementation on Glucose (mg/dL) levels of albino wistar male

rats

80.00

90.00

100.00

110.00

120.00

130.00


Glu

co

se (

mg

/dL

)


Level of significance (Treatments: <0.05ns, Intervals: >0.05ns)Level of significance (Treatments: >0.05ns, Intervals: >0.05ns)

Figure 4. 54: Effect of PoPx supplementation on Glucose (mg/dL) levels of albino wistar male

rats

150

Table 4.25: Analysis of variance for blood chemistry

ns = non-significant, * = significant, ** = highly significant

Variables Triglycerides Cholesterol HDL LDL Uric acid Creatinine Bilirubin Total

protein

Albumin ALT ALP AST Glucose

PoP

Days 17.29* 6.8ns 0.40ns 5.89* 0.007* 0.005** 0.002** 0.0002ns 0.0002ns 0.759ns 2.154ns 1.047ns 0.16ns

Treatment 93.19** 24.4* 1.27ns 6.48** 0.008** 0.003** 0.001** 0.002ns 0.003ns 1.799ns 11.924* 0.327ns 0.92ns

Days *

Treatment

9.47* 2.08ns 0.10ns 0.35ns 0.001** 0.0008* 0.0002* 0.0001ns 0.0002ns 0.496ns 0.094ns 0.326ns 0.76ns

PM

Days 43.843 0.44ns 0.082ns 3.362ns 0.002ns 0.002ns 0.001ns 0.0004ns 0.0006ns 0.074ns 0.881ns 0.267ns 0.46ns

Treatment 17.982 10.89ns 3.720ns 7.067ns 0.006ns 0.000ns 0.001ns 0.007ns 0.002ns 1.144ns 13.334* 0.560ns 2.26ns

Days *

Treatment 2.696 0.34ns 0.306ns 0.059ns 0.0003ns 0.000ns 0.000ns 0.0001ns 0.00006ns 0.287ns 0.097ns 0.169ns 0.45ns

PoPx

Days 36.69** 19.96* 0.57ns 2.76ns 0.008** 0.005** 0.004** 0.001ns 0.00007ns 1.061ns 1.731ns 1.853ns 0.07ns

Treatment 126.37** 16.68* 2.56* 9.47** 0.009** 0.006** 0.002** 0.0002ns 0.002ns 4.241** 14.420* 10.787** 2.29ns

Days *

Treatment

18.05* 6.19ns 0.18ns 0.48ns 0.002** 0.0007* 0.0006* 0.0005ns 0.0001ns 0.709ns 0.095ns 0.611ns 0.83ns

151

4.16 Rats Hematology

Analysis of hematological parameters is an essential part of preclinical as well as

clinical assessment in safety evaluation of conventional and innovative therapeutics of

human concern. Hematology is generally performed to assess target oriented toxicity of

pharmaceutical preparations, food additives, agrochemicals, medical and surgical tools.

Hematological assessment being carried out in safety assessment studies defines blood cells

response towards the agents administrated in terms of bone marrow impairment and expected

damage to the blood cells (Bloom 1993; James 1993).

Present study did not reveal any harmful or hazardous effect of feeding PoP, PM and

PoPx supplemented diets to normal rats on hematological parameters during the entire study

period. The hematological analysis of the study groups (PoP, PM and PoPx) revealed dietary

supplementation of pomegranate peel and its various fractions at different levels to not

generate toxic response on feeding for a period of 56days. Moreover, minimum to maximum

supplementation of PoP, PM and PoPx was also found to yield hematological results

comparable to those of control animals. Hence, supplementation response of pomegranate

peel and its derived fractions may be referred as safe for consumption at the tested doses i.e.

2.0 – 6.0% for PoP and PM and 0.25 – 1.0% for PoPx.

4.16.1 Effect of feeding PoP,PoPx and PM on white blood cells of Albino wistar rats

Ingestion of diets supplemented with pomegranate peel and its derived fractions i.e.

the meal and hydro-alcoholic extracts revealed a non-significant change in white blood cells

(WBCs) counts of the experimental animals. Analysis of variance (Table 4.35) indicates a

non-significant effect for the treatment combinations, feeding intervals and interaction

among these variables when compared with the control on WBCs indices of the PoP, PM and

PoPx feed rats.

Means comparison of the WBCs counts of the experimental rats at intervals of 0, 28

and 56 days revealed a non- significant (p>0.05) effect of time on WBCs counts (Table 4.26).

A similar trend was noticed for the supplementation levels with PoP, PM and PoPx as none

of the study groups had shown any change in WBCs counts. Comparison for the feeding

152

intervals among different rats’ groups exhibited that PM supplementation for 56 days had

reduced WBCs count by 0.63% (p>0.05) while the rats supplied with PoP and PoPx

supplemented diets did not depict any significant effect of feeding time. The variability in

WBCs counts had been little enough i.e. 0.14% and 0.28% for PoP and PoPx supplemented

group respectively to suggest any deleterious effect of PoP and PoPx supplementation,

respectively on WBC counts. The data further suggest phenolic rich PoP and PoPx to offer

comparatively lower rate of reduction in WBC counts. The findings of current work are in

agreement with those observed by Tasaki et al. (2008) who demonstrated 1.25%

supplementation of ellagic acid – notable hydrolysable tannin of pomegranate to produce

~0.9% increase in WBCs contents of the male rats. WBC counts identified in current study

are also comparable to the results presented by Sen et al. (2014) where oral feeding of

pomegranate extracts to the rats model was recorded with WBCs ~5500.

Table 4.26: Effect of PoP, PM and PoPx supplementation on white blood cells counts of

albino wistar rats

Group Treatments Mean ± SD (0

Days)

Mean ± SD (28

Days)

Mean ± SD (56

Days)

Mean

PoP

Control 5483.3±76.38a 5466.7±76.38a 5526.7±155.35a 5492.2a

2.00% 5476.7±135.77a 5480.0±121.24a 5436.7±63.51a 5464.4a

4.00% 5503.3±92.92a 5486.7±15.28a 5466.7±49.33a 5485.6a

6.00% 5496.7±35.12a 5503.3±28.87a 5500.0±0.00a 5500.0a

5490.0a 5484.2a 5482.5a

PM

Control 5483.3±76.38a 5466.7±76.38a 5526.7±155.35a 5492.2a

2.00% 5550.0±45.83a 5543.3±100.66a 5510.0±17.32a 5534.4a

4.00% 5563.3±55.08a 5523.3±66.58a 5513.3±47.26a 5533.3a

6.00% 5560.0±40.0a 5510.0±34.64a 5466.7±55.08a 5512.2a

5539.2a 5510.8a 5504.2a

PoPx

Control 5483.3±76.38a 5466.7±76.38a 5526.7±155.35a 5492.2a

0.25% 5556.7±95.04a 5540.0±69.28a 5526.7±40.41a 5541.1a

0.50% 5583.3±55.07a 5540.0±34.64a 5533.3±85.05a 5552.2a

1.00% 5553.3±50.33a 5533.3±32.15a 5526.7±25.17a 5537.8a

5544.2a 5520.0a 5528.3a

Means sharing same lettering in rows and columns are statistically non-significant (p>0.05)

153

4.16.2 Red Blood Cells

Data pertaining to the levels of red blood cells (RBCs) of experimental rats on

feeding pomegranate peel, meal and extracts fraction are presented in Table 4.27.

Supplementation of pomegranate peel and its fractions in the rats’ diets have been shown to

render a significant effect on RBCs of the albino wistar rats (Table 4.35). Likewise, feeding

time of supplemented diets had also indicated a significant effect on RBCs levels of the rats

along with significant (p<0.05) interaction between the treatment and study interval.

The results pertaining to variation in RBCs levels among the experimental rats as a

result of feeding diets supplemented with PoP, PM and PoPx depicted improvement in RBCs

levels such as PoP supplementation resulted in 6.0% increase in RBCs in a period of 56 days.

Similarly, a comparison of the means of values for RBCs counts among rats fed with PM and

PoPx supplemented diets also presented a slight increase in the RBCs i.e. 0.6 and 0.7%,

respectively.

Comparison for the level of supplementation in a specific group indicated significant

improvement in RBCs counts with increments in PoP, PM and PoPx supplementation.

Maximum improvement in RBCs counts of the rats were identified in PoP supplemented

group at 6.0% supplementation which resulted in an increase in RBCs counts from 6.671 –

6.837 million/µl suggesting approximately 2.5% increase in comparison with the control.

The variation in RBCs level was noticed in a dose dependent manner as the highest levels of

supplementation for PoPx and PM supplemented groups indicated 0.55% (6.671 – 6.708

million/µl) and 0.8% (6.671 – 6.724 million/µl) increase in RBCs counts as compared to the

control. A retrospective study on whole fruit extracts supplementation at 2000mg/kg/day for

a period of 28 days was shown to produce 9.36% increment in RBCs of the experimental rats

(Bhandary et al. 2013). The findings of current study delineate lower tendency of PoP, PM

and PoPx to increase RBCs in rats’ model probably due to lower elemental iron contents of

the fruit and waste fraction as has been defined in mineral composition of pomegranate peel

powder of Alipuri cultivar.

154

Table 4.27: Effect of PoP, PM and PoPx supplementation on Red blood cells counts

(millions/µl) of albino wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 6.660±0.004g 6.668±0.003g 6.680±0.017g 6.671d

2.00% 6.667±0.015g 6.797±0.021e 6.825±0.005d 6.763c

4.00% 6.723±0.025f 6.843±0.021cd 6.873±0.006b 6.813b

6.00% 6.708±0.019f 6.870±0.02bc 6.933±0.015a 6.837a

6.691c 6.795b 6.828a

PM

Control 6.660±0.004e 6.668±0.003e 6.680±0.017d 6.671c

2.00% 6.675±0.005de 6.691±0.005c 6.712±0.003b 6.693b

4.00% 6.671±0.002de 6.714±0.004b 6.727±0.004a 6.704a

6.00% 6.673±0.003de 6.721±0.006ab 6.729±0.004a 6.708a

6.671c 6.699b 6.712a

PoPx

Control 6.660±0.004f 6.668±0.003ef 6.680±0.017d 6.671c

0.25% 6.665±0.003f 6.676±0.005def 6.699±0.006c 6.680b

0.50% 6.668±0.003ef 6.678±0.003de 6.709±0.006c 6.685b

1.00% 6.671±0.004def 6.733±0.007b 6.766±0.008a 6.724a

6.668c 6.689b 6.714a


155

4.16.3 Hemoglobin contents

Hemoglobin contents of the albino wistar rats fed PoP, PM and PoPx supplemented

diets at various dose levels are presented in Table 4.28. The analysis of variance (Table 4.35)

reveals a significant effect of supplementation and time of exposure on hemoglobin levels of

rats fed PoP, PM and PoPx supplemented diet. Moreover, a positive and significant

interaction among feeding time and treatment combinations as variables was observed in the

present study.

Dietary administration of pomegranate peel and the bagasse (20g/d) to rams for a

period of six weeks have been reported to improve hemoglobin concentration from 8.53 –

12.4g/dL (Imad Hamad et al. 2016). Comparison of means for the hemoglobin levels of rats

fed PoP, PM and PoPx supplemented diet exhibited significant effect of feeding interval on

hemoglobin contents of the albino wistar rats (Table 4.28). The study further indicates PoP to

have an ability to improve hemoglobin contents of the rats at 3.79% during 0 – 56 days of

feeding whereas exposing rats to PM and PoPx supplemented diet for a period of 56days

improved hemoglobin contents by 2.41 and 2.95%, respectively. In comparison with the

hemoglobin contents of the control rats i.e. 11.72g/dL, rats fed on 1.0% PoPx and 6.0% PM

were noticed with 11.10 and 11.11mg/dL hemoglobin, respectively. Increasing

supplementation levels of PoP, PM (2.0 – 4.0%) and PoPx (~0.5%) in rat’s diet resulted in a

significant increase in hemoglobin contents while further increase in supplementation of all

tested fractions did not significantly improve hemoglobin contents of rats under

investigation.

No treatment in all three groups (PoP, PM and PoPx) induced any toxicity in terms of

reduction in hemoglobin contents. Within the treatments, maximum hemoglobin contents

were noticed in PoP supplemented groups where 2 – 6.0% supplementation of PoP improved

hemoglobin levels from 11.233 – 11.433g/dL. The findings of the present work are therefore

suggestive of non-toxicological effect of any of pomegranate peel fraction on hemoglobin

contents at the highest supplementation level (6.0%). Oral administration of pomegranate

extracts (225mg/kg/b.w) with carvacrol to albino wistar rats for a period of seven days was

shown to depict 9.95g/dL hemoglobin contents in rats i.e. approximately 13% lesser than the

values noted in normal rats (Sen et al. 2014). However, our workshows PoP, PM and PoPx

156

(estimated maximum consumption - 995mg/kg b.w./day) to depictnon inhibitory properties

on hemoglobin contents at their maximum supplementation levels i.e. 6.0% for PoP and PM

and 1.0% for PoPx.

Table 4.28: Effect of PoP, PM and PoPx supplementation on hemoglobin (g/dL) of albino

wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD (56

Days)

Mean

PoP

Control 11.667±0.06abcd 11.767±0.06ab 11.733±0.06abc 11.722a

2.00% 11.133±0.05f 11.267±0.06f 11.300±0.10ef 11.233c

4.00% 11.133±0.21f 11.500±0.10de 11.633±0.06bcd 11.422b

6.00% 10.900±0.20g 11.533±0.06cd 11.867±0.25a 11.433b

11.208c 11.517b 11.633a

PM

Control 11.667±0.06a 11.767±0.06a 11.733±0.06a 11.722a

2.00% 10.800±0.10e 10.900±0.10e 10.933±0.15de 10.878c

4.00% 10.933±0.15de 11.083±0.08cd 11.233±0.06bc 11.083b

6.00% 10.833±0.15e 11.100±0.10cd 11.400±0.10b 11.111b

11.058c 11.213b 11.325a

PoPx

Control 11.667±0.06a 11.767±0.06a 11.733±0.06a 11.722a

0.25% 10.783±0.08f 11.017±0.13e 11.15±0.05cd 10.983c

0.50% 10.850±0.05f 11.083±0.08de 11.250±0.05bc 11.061b

1.00% 10.833±0.06f 11.167±0.06cd 11.300±0.05b 11.100b

11.033c 11.258b 11.358a


157

4.16.4 Hematocrits

Hematocrits (HCT) are a measure of red blood cells volume as compared to the

volume of blood that varies with gender and specie (Billett 1990; Nemeth et al. 2009). HCT

percentage of blood samples derived from albino wistar rats at 0, 28 and 56 days of feeding

supplemented diets are presented in Table 4.29. Analysis of variance for HCT contents

(Table 4.35) revealed a significant (p<0.05) effect of feeding interval on HCT percentage

while non-significant (p>0.05) effect of increasing level of PoP, PM and PoPx

supplementation in the diets was observed in HCT percentage of the male albino wistar rats.

Data presented in Table 4.42 for HCT contents revealed maximum supplementation

of PoP, PM and PoPx to present 35.20, 34.77 and 34.93% HCT contents, respectively as

compared to the control i.e. 34.70% at 56th day of feeding. Comparatively, higher rate of

increase (~2.0%) in HCT percentage was noticed in rats fed on PM supplemented diet

whereas PoP supplementation for a period of 56 days resulted in ~1.5% HCT increment.

Significant but relatively lower rate of improvement in HCT percentage i.e. 1.15% was seen

in rats fed on PoPx supplemented diet. Similarly, comparison of means for the treatments

within the groups revealed non-significant effect of increasing levels of PoP supplementation

on HCT percentage (Table 4.29).

Our results indicated that PoP, PM and PoPx supplementation of rats diets did not

negatively alter HCT contents of the experimental animals. Among various treatments of

three tested groups, supplementation at any proposed level did not show any abnormal

change in HCT contents among the tested rats. Baseline value for hematocrits in 19 – 21

week aged male wistar rats were reported in a range of 36 – 46% by Charles River

Laboratories (1998). Interpretation of findings of this research indicate relatively lower level

of HCT in control animals that was gradually built up with PoP, PoPx and PM

supplementation thus depicting a positive role of pomegranate peel and associated fractions

in improving HCT contents of experimental rats.

158

Table 4.29: Effect of PoP, PM and PoPx supplementation on hematocrits (HCT %) of albino

wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD (56

Days)

Mean

PoP

Control 34.667±0.06bc 34.733±0.12bc 34.700±0.10bc 34.700ab

2.00% 34.767±0.15cd 35.433±0.06bc 35.600±0.30b 35.267b

4.00% 34.433±0.06d 34.883±0.08b 35.133±0.21a 34.817a

6.00% 34.100±0.20e 34.767±0.15bc 35.200±0.10a 34.689ab

34.442c 34.762b 34.963a

PM

Control 34.667±0.06c 34.733±0.12c 34.700±0.10c 34.700b

2.00% 34.567±0.06e 34.667±0.15b 34.817±0.10a 34.683a

4.00% 34.167±0.15e 34.367±0.15d 34.633±0.06c 34.389c

6.00% 34.133±0.06e 34.400±0.10d 34.767±0.15bc 34.433c

34.283c 34.608b 34.975a

PoPx

Control 34.667±0.06bcd 34.733±0.12b 34.700±0.10bc 34.700a

0.25% 34.207±0.10fg 34.310±0.04f 34.483±0.06e 34.333c

0.50% 34.097±0.05g 34.337±0.07f 34.590±0.07cde 34.341c

1.00% 34.157±0.06g 34.540±0.06de 34.933±0.15a 34.543b

34.282c 34.480b 34.677a


159

4.16.5 Platelets counts

Effect of PoP, PM and PoPx supplementation on platelets counts of albino wistar

rats is presented in Table 4.30. The analysis of variance for platelets counts as depicted in

Table 4.35 revealed a significant effect of supplementation and feeding intervals on the rats’

platelets count. Similarly, a significant (p<0.05) interaction was noticed among the two

variables i.e. feeding interval and supplementation level on platelet counts of the

experimental animals.

Feeding rats with PoP, PM and PoPx for a period of 56days resulted in 2.7%, 1.56%

and 3.06% improvement in platelets counts respectively. Maximum platelets counts were

seen in rats’ fed with PoP supplemented diet where the mean value at 56th day was recorded

to be 571.25 (thousand counts) as compared to 553.3 (thousand counts).

Significant effect of supplementation levels was noticed on platelets counts of rats fed

various fractions of pomegranate peel. Table 4.30 shows 6.0% supplementation of PoP and

PM and 1.0% supplementation of PoPx increased platelets counts of the respective

experimental group by 3.79%, 1.52% and 2.91%, respectively. Virtually increasing trend in

platelets counts was identified from this study with increasing supplementation levels.

Effect of PoP and PoPx supplementation on platelet counts of male wistar rats are in

agreement with the findings of Patel et al. (2008) where 600mg/kg/day oral feeding of

extracts for a period of 90days was recorded to enhance platelet counts up to 4.8%. Oral

administration of pomegranate whole fruits and seed extracts at 2000mg/kg/day have also

been reported to increase platelets in normal rats i.e. 11.8% from 314 to 356 (×103 per µl)

(Bhandary et al. 2013).

160

Table 4.30: Effect of PoP, PM and PoPx supplementation on platelets (thousands) counts of

albino wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 553.0±2.00f 553.5±0.50f 553.3±0.58f 553.3d

2.00% 550.0±5.00f 559.0±3.61e 572.3±3.06cd 560.4c

4.00% 559.0±3.61e 570.0±2.00d 576.7±1.53bc 568.6b

6.00% 563.3±2.52e 577.0±1.00b 582.7±2.08a 574.3a

556.33c 564.88b 571.25a

PM

Control 553.0±2.00d 553.5±0.50d 553.3±0.58d 553.3c

2.00% 559.3±1.53c 560.2±2.02c 564.5±1.50b 561.3ab

4.00% 554.2±2.75d 561.2±0.76c 565.0±1.38b 560.1b

6.00% 550.5±1.32e 565.8±0.76b 568.7±0.29a 561.7a

554.25c 560.17b 562.88a

PoPx

Control 553.0±2.00e 553.5±0.50e 553.3±0.58e 553.3d

0.25% 554.3±2.08e 563.0±3.00d 569.7±1.53c 562.3c

0.50% 553.3±2.08e 565.7±4.04d 575.3±2.08b 564.8b

1.00% 552.8±1.61e 572.3±3.06bc 583.0±2.00a 569.4a

553.38c 563.62b 570.33a


161

4.16.6 Lymphocytes

Lymphocytes with their three subtypes including natural killer (NK) cells, T-cells or

thymus cells and B-cells or bursa cells are classes of white blood cells found in lymphatic

system. Presence of lymphocytes in lymphatic system defines innate immune system, cell

mediated immunity and humoral immunity of the body. Baseline values of lymphocytes for

wistar rats aged 19 – 21weeks have been documented in a range of 70 – 99% with a mean

value of 82% (Charles River Laboratories 1998).

Lymphocytes counts of albino wistar rats fed pomegranate peel, meal and peel

extracts are given in Table 4.31. Our results indicate a significant effect of PoP

supplementation and feeding time of PoP supplemented diets on blood lymphocytes contents

of rats. Significant effect of feeding intervals was also observed among rats provided with

PoPx supplemented diet whereas non-significant effect of supplementation, feed interval and

interaction among variables was noticed from the lymphocytes of PM fed rats.

Means comparison for effect of PoP, PM and PoPx supplementation in rats diet

revealed significant increase in lymphocytes percentage during first phase of the feeding

period i.e. 0 – 28th day whereas extending rats feeding program with PoP, PM and PoPx

supplemented diets up to 56days did not reflect any significant change in blood lymphocytes

contents. Maximum increment in lymphocytes contents was noticed in PoP feed group where

lymphocytes contents in this study group were significantly declined by1.2% whereas rate of

lymphocytes increment for PM and PoPx fed rats was observed to be 0.7% and 0.9%,

respectively.

Pomegranate extracts evaluated for their suspected toxicity through oral feeding have

been previously reported to increase lymphocytes contents from 81.7 – 83.20% in rats

exposed to 600mg/ kg body weight extracts for a period of 90 days (Patel et al. 2008). These

researchers have demonstrated relatively lower levels of lymphocytes i.e. 82.49% in rats fed

1.0% pomegranate fruit extracts - a relatively higher dose of the extracts than administrated

in the current study suggesting non-toxic effects of PoP and associated fractions at highest

levels of supplementation i.e. 6.0% for PoP and PM and 1.0% for PoPx.

162

Table 4.31: Effect of PoP, PM and PoPx supplementation on lymphocytes (%) of albino

wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 81.76±0.26d 82.62±0.87abcd 82.05±1.21cd 82.14b

2.00% 82.17±0.29bcd 82.55±0.49abcd 83.38±0.93a 82.70ab

4.00% 82.69±0.85abcd 83.17±0.73abc 83.60±0.04a 83.15a

6.00% 81.86±0.75d 82.74±0.52abcd 83.33±0.52ab 82.64ab

82.121b 82.769a 83.090a

PM

Control 81.76±0.26a 82.62±0.87a 82.05±1.21a 82.14a

2.00% 81.86±0.80 82.09±0.60 82.28±0.45 82.08a

4.00% 81.97±0.34 82.20±0.34 82.59±0.30 82.25a

6.00% 81.84±0.79 82.21±0.28 82.75±0.39 82.27a

81.856b 82.281ab 82.414a

PoPx

Control 81.76±0.26b 82.62±0.87ab 82.05±1.21ab 82.14a

0.25% 81.77±1.08b 82.19±0.57ab 82.57±0.30ab 82.18a

0.50% 81.61±0.40b 81.95±0.21ab 82.54±0.76ab 82.03a

1.00% 81.93±0.18ab 82.59±0.17ab 82.93±0.68a 82.49a

81.77b 82.339a 82.522a


163

4.16.7 Neutrophils counts

Neutrophils being integral part of innate and adoptive immunity play a significant

role in resolving and aggravating pathogenesis including those of chronic inflammation,

autoimmune disorders and various forms of cancers. Neutrophils homeostasis is highly

important to eliminate the risk of neutropenia and neutrophilia to avoid the risks of infections

and healthy cells damages (Summers et al. 2010; Jaillon et al. 2013). Baseline values of

neutrophils for wistar rats aged 19 – 21weeks are reported in a range of 1– 29% with a mean

value of 15% (Charles River Laboratories 1998).

Present study revealed a non-significant (p>0.05) effect of feeding time and the

supplementation level on blood neutrophils contents among the tested rats groups (Table

4.35). Contrary to PoPx and PM, feeding rats a diet supplemented with PoP at various levels

indicated significant (p<0.05) effect of PoP supplementation on mean neutrophil contents as

compared to control. Despite, modest change in blood neutrophils of the rats (study group) as

compared to the control, the study did not show any sign of neutropenia probably due to a

balance ratio of lymphocytes and neutrophils in the experimental animal. Moreover, the

observed levels were quite in range prescribed for male albino rats of the same age group.

Differential percentage of neutrophils in albino wistar rats fed on pomegranate peel, meal and

peel extracts are given in Table 4.32.

The study further depicts PoP, PM and PoPx supplementation in the basal diet of rats

to show non-significant effect of supplementation on blood neutrophil contents. Among

groups, treatments comparison further reveals non-significant effect of increasing

supplementation levels (from 2.0 – 6.0%) on neutrophil contents of PoP and PM

supplemented diet fed rats. Feeding rats with diets carrying 1.0% PoPx rendered significant

decline i.e. 13.819 to 13.003% in neutrophil contents over the time (56days). Abrupt change

in neutrophils contents of rats fed with PoPx supplemented diet was also an indicator of non-

treatment factor for this significant change. Similar trend as has been reported in current

study, was reported by Patel et al. (2008) during acute and sub-chronic safety evaluation of

pomegranate fruit extracts revealing ~7.0% loss in neutrophil counts at 200mg/kg/day oral

administration of extracts whereas the rate of reduction was reduced to up to 2% at

600mg/kg/day dose.

164

Table 4.32: Effect of PoP, PM and PoPx supplementation on neutrophils (%) of albino

wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 13.986±0.06a 13.724±0.06ab 13.748±0.06ab 13.819a

2.00% 13.327±0.06abc 13.194±0.06abc 12.259±0.10c 12.927b

4.00% 13.025±0.21abc 12.939±0.10abc 12.805±0.06bc 12.923b

6.00% 13.521±0.20ab 13.083±0.06abc 12.727±0.25bc 13.110b

13.465a 13.235a 12.885a

PM

Control 13.986±0.06a 13.724±0.06a 13.748±0.06a 13.819a

2.00% 13.575±0.10 13.412±0.10 13.309±0.15 13.432ab

4.00% 13.300±0.15 13.274±0.08 13.178±0.06 13.251b

6.00% 13.248±0.15 13.248±0.10 13.169±0.10 13.222b

13.527a 13.415a 13.351a

PoPx

Control 13.986±0.06a 13.724±0.06ab 13.748±0.06ab 13.819a

0.25% 13.492±0.08ab 13.176±0.13ab 13.025±0.05ab 13.231ab

0.50% 13.491±0.05ab 13.478±0.08ab 13.255±0.05ab 13.408ab

1.00% 13.328±0.06ab 12.954±0.06ab 12.726±0.05b 13.003b

13.574a 13.333a 13.189a


165

4.16.8 Mean Corpuscular Volume ( MCV)

Mean corpuscular volume is referred to be a measure of average volume of the red

blood cells and the abnormally higher volume of the cells are recorded with either the

deficiency of Vitamin B12 or folates. MCV of the blood samples collected from control and

treated rats has been presented in Table 4.33. The analysis of variance reveals significant

effect of feeding interval and supplementation on the MCV of the red blood cells in rats fed

on PoPx and PM (Table 4.35). Feeding interval was identified to non-significantly affect

MCV in rats fed on PoP supplemented diet.

Comparison of means for MCV has been presented in Table 4.33. The study revealed

that feeding time of supplemented diets exerted a non-significant effect on MCV of the rats

fed on PoP supplemented diet whereas ~1.4% increment in MCV was observed in rats

provided with PM supplemented diet for a period of 56 days. The data presented in this study

further indicate PoPx supplementation in rats diet produced significant effect of

supplementation on MCV in 28 days of feeding while non-significant (p>0.05) change in

MCV was witnessed on sub-chronic (56 days) feeding.

Our results further validated significant decline in MCV value by increasing dietary

concentration of PoP i.e. 2.0 – 6.0% of the rats diet. However; gradual increment in PoPx

concentration in rats; diet did not reflect any significant change in MCV. Contrary to the PoP

and PoPx group, rats fed with 2.0 – 6.0% PM supplemented diet were found to carry ~1.8%

higher MCV as compared to the control at maximum supplementation level i.e. 6.0%.

PoPx at 0.25 – 1.0% supplementation induced a negligible decline in red blood cells

volume as compared to PoP thereby suggesting a non-toxic effect of PoPx supplementation

at maximum supplementation levels (1.0%).

166

In comparison with the toxicological safety assessment of pomegranate fruit extracts

carried out by Patel et al. (2008), PoPx at its maximum supplementation level i.e. 1.0% or

~995mg/kg/day anticipated 1.2% mean cell volume loss as compared to 15.9% in 90 days

oral feeding of 600 mg/kg/day. Non-significant effect of 1.0% pomegranate extracts enriched

diet on MCV of Olive flounder were also witnessed by Harikrishnan et al. (2012). Increase in

MCV volume may result hypoxic conditions, macrocytic anemia or osmotic stress. The

findings from current study did not witness any such change in MCV thus referring PoPx as a

safer ingredient to be incorporated in food preparations.

Table 4.33: Effect of PoP, PM and PoPx supplementation on mean corpuscular volume of

albino wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 52.01±0.08a 52.09±0.18a 51.95±0.08a 52.014a

2.00% 52.15±0.31a 52.13±0.08a 52.16±0.46a 52.184a

4.00% 51.22±0.12b 50.97±0.05bcd 51.12±0.29bc 51.102b

6.00% 50.83±0.35bcd 50.61±0.37d 50.77±0.04cd 50.736c

51.551a 51.451a 51.498a

PM

Control 52.01±0.08b 52.09±0.18b 51.95±0.08b 52.014b

2.00% 51.22±0.12e 51.19±0.21b 51.48±0.13a 51.297a

4.00% 51.15±0.24de 51.18±0.21e 51.67±0.11cd 51.333c

6.00% 51.18±0.10e 52.21±0.13e 53.34±0.21c 52.243c

51.389c 51.667b 52.109a

PoPx

Control 52.01±0.08a 52.09±0.18a 51.95±0.08a 52.014a

0.25% 51.32±0.17def 51.39±0.06cde 51.47±0.09bcd 51.396b

0.50% 51.14±0.08f 51.42±0.09bcde 51.56±0.10bc 51.369b

1.00% 51.20±0.08ef 51.30±0.14def 51.63±0.28b 51.376b

51.416b 51.550a 51.651a


167

4.16.9 Mean Corpuscular Hemoglobin Concentration

Mean corpuscular hemoglobin concentration (MCHC) is a measure of hemoglobin

concentration in RBCs given packed cell volume. Abnormal decline in MCHC value

indicates hypochromic or microcytic anemia whereas the elevated MCHC levels are referred

hyperchromic that might be an indicator of sickle cell anemia.

Significant effect of PoP, PoPx and PM supplementation and the feeding interval was

recorded on MCHC value of albino wistar male rats. Mean comparison of MCHC for various

groups and treatments for a period of 56 days indicates 1.06 – 3.93% increase in MCHC of

the tested rats as compared to the control thus referring sub-chronic feeding of the

pomegranate peel and derived fractions to exert least to no toxic effect on MCHC volume

(Table 4.34). The data presented illustrated that PoP and PM supplementation in rats diet

between 4.0 – 6.0% and PoPx supplementation between 0.5 – 1.0% had a non-significant

effect on MCHC.

A toxicological sub-chronic and acute safety assessment study on pomegranate fruit

extracts reveals oral administration of 600mg/kg/b.w for a period of 90 days to increase

MCHC volume by 17.5% (Patel et al. 2008) whereas, the current study reveals 1.0% PoPx

supplementation to impart ~4.1% increment in MCHC volume of the male rats. Seemingly,

pomegranate peel supplementation ~4.0% in lambs diet have also been reported to increase

MCHC by approximately 3.0% (Ramzi 2016) that is in agreement with the findings of

current study.Another study reported from Harikrishnan et al. (2012) also suggest 1.0%

dietary supplementation of pomegranate extracts in olive flounder fish for a period of 4

weeks to offer a significant increase in MCHC as has been identified in current study.

168

Table 4.34: Effect of PoP, PM and PoPx supplementation on mean corpuscular volume of

albino wistar rats


Days)

Mean ± SD (28

Days)

Mean ± SD

(56 Days)

Mean

PoP

Control 22.43±0.08b 22.59±0.17b 22.59±0.14b 22.537a

2.00% 21.35±0.22c 21.61±0.08c 21.67±0.34c 21.542b

4.00% 21.74±0.36c 22.56±0.21b 22.76±0.08b 22.353a

6.00% 21.44±0.44c 22.79±0.10b 23.37±0.50a 22.536a

21.741b 22.388a 22.596a

PM

Control 22.43±0.08ab 22.59±0.17a 22.59±0.14a 22.537a

2.00% 21.10±0.16fg 20.88±0.22g 20.50±0.34h 20.826c

4.00% 21.35±0.26ef 21.65±0.21de 21.82±0.15cd 21.606b

6.00% 21.18±0.33fg 21.69±0.19de 22.06±0.25bc 21.644b

21.515b 21.702a 21.743a

PoPx

Control 22.43±0.08a 22.59±0.17a 22.59±0.14a 22.537a

0.25% 21.01±0.11f 21.44±0.26de 21.66±0.13bcd 21.370c

0.50% 21.22±0.06ef 21.56±0.16cd 21.82±0.11b 21.532b

1.00% 21.16±0.09f 21.77±0.16bc 21.89±0.19b 21.605b

21.455c 21.837b 21.990a


169

Table 4.35: Analysis of variance for hematological parameters of albino wistar normal male rats feed on PoP, PM and PoPx

supplemented diet for 0, 28 and 56days

Variables WBC RBC HGB HCT Platelets Lymph Neutrophil MCV MCHC

PoP

Days 186.11ns 0.061** 0.579** 0.828** 672.215** 2.926* 1.023ns 0.030ns 2.387**

Treatment 2096.3ns 0.049** 0.366** 0.036ns 765.137** 1.531* 1.629* 4.279** 2.027**

Days

Treatment

1826.9ns 0.007** 0.126** 0.174** 80.623** 0.355ns 0.189ns 0.024ns 0.514**

Error 7194.4 0.000 0.016 0.018 6.910 0.486 0.488 0.060 0.071

PM

Days 4144.4ns 0.005** 0.215** 1.437** 233.843** 1.017ns 0.095ns 1.579** 0.177*

Treatment 3610.2ns 0.002** 1.194** 0.644** 139.556** 0.074ns 0.681ns 2.068** 4.404**

Days *

Treatment

2918.5ns 0.001** 0.039** 0.346** 55.679** 0.204ns 0.013ns 0.748** 0.295**

5322.2 0.000 0.011* 0.013 2.145 0.389 0.321 0.026 0.049

PoPx

Days 1808.3ns 0.006** 0.333** 0.468** 875.299** 1.842* 0.455ns 0.167** 0.909**

Treatment 6306.5ns 0.005** 1.044** 0.280** 413.130** 0.336ns 1.073ns 0.904** 2.496**

Days *

Treatment

1512.0ns 0.001** 0.024** 0.076** 123.012** 0.214ns 0.036ns 0.051* 0.058*

5577.8ns 0.000 0.005 0.007 5.097 0.430 0.384 0.018 0.022

ns = non-significant, * = significant, ** = highly significant

170

Summary and Recommendations

Pomegranate peel (PoP) and pomegranate peel extracts (PoPx) have been shown to have

substantial potential for being key ingredients in ethnopharmacological formulations of almost

all cultures of the world. Owing to be a rich source of inexpensive vital health component, PoP is

widely utilized as a curative agent for certain common health disorders including inflammation,

diarrhea, dysentery, mouth plaques and intestinal infections. PoP and the products derived

thereof including PoPx provide numerous health benefits in the form of nutraceuticals and bio-

preservatives. Despite these remarkable functional properties, the peel fraction comprising up to

30% of the fruit weight still goes directly into the waste stream of juice manufacturing industries.

This situation calls for investigation to be directed to unveil food value of PoP and its utilization

as disease preventive, health promoting bio-ingredient and a food system stabilizer.

In vitro and in vivo studies have validated exceptional antioxidant potential of the peel

and its preventive role against some critical diseases like prostrate, colon and liver cancers,

stomach ulcers, cardiovascular diseases and digestive disorders revealing their cytoprotective

potential and inhibitory effects. PoP has also been observed to be able to serve as preventive

strategy against some human carcinomas and, to some extent, a therapeutic agent.

Biomolecules available in PoP and PoPx hold strong food relevance suggesting their

application as substitutes of synthetic food additives. Some studies report the ingestion of

pomegranate and its peel fractions in the form of pills, capsules and gels as conventional

treatment regimens against certain diseases. Utilization of pomegranate peel and its extract in

defined concentrations in various organoleptically acceptable food preparations, as effective

supplements and food additives, might open new avenues for scientific research in the realm of

food science and nutrition. Incorporation of PoP or its fractionated phytochemicals could

practically serve an equal role as bio-preservative and health promoting agent. Utilization of PoP

as a reservoir of valuable therapeutic agents may act as food preservatives, stabilizers,

supplements, probiotics and quality enhancement agents in addition to be a pragmatic disease

preventive approach. However, careful exploration to spotlight the efficacy of PoP and PoPx as

biologically safe ingredients for consumption as food or a part of food system, stability of active

ingredients under various food processing conditions and sensorial features of the finished

consumer goods, is direly needed to fully exploit the waste of this heavenly fruit.

171

Present research aimed at exploring food value of PoP and its derived extracts. These

ingredients have shown remarkable functional characteristics with tremendous potential to be

used in various food recipes. The study exhibited a significant variability in biological properties

of PoP derived from indigenous white rind cultivar “Alipuri” as compared with globally

distributed cultivars of pomegranate. Hydro-alcoholic extraction of peel fractions was found to

exhibit a significant correlation among type of solvent, extracts recovery and phenolics yield.

Acetone extracts manifested increased ability for inhibition of oxidation with greater urease

activity and antimicrobial potential as compared to methanolic and ethanolic extracts. Higher

levels of total phenolics (427.19mgGAE/g) recovered from hydro-alcoholic extracts showed a

significant (P<0.05) ferric reducing antioxidant power (FRAP) and 1, 1-diphenyl-2-

picrylhydrazyl (DPPH) free radicals scavenging activity.

Results obtained from current study manifested that PoPx inhibited urease activity by

97.9% and IC50 for all extracts was estimated to be in a range of 30 – 44.4µM. Potent inhibitory

effects of PoP extracts were observed against Bacillus subtilis, Staphylococcus aureus,

Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium and Aspergillus niger and

minimum inhibitory concentration (MIC) was recorded between 0.25 – 0.89mg/mL. The study

indicates that PoPx of the white rind cultivar of pomegranate is a potential source of active

biomolecules that could further be explored as a source of bioactive compounds and therapeutic

agents against various ailments. All extracts, at their highest tested concentration, were found to

be non-toxic in the brine shrimps lethality assay. Findings of the present study are not consistent

with several retrospective studies in terms of yellow / white rind pomegranate cultivars being

weak source of oxidation inhibitory compounds as compared to the red rind cultivars. The rind of

the indigenous pomegranate cultivar has been found to be exceptionally effective source of

antioxidant agent. The study further suggests that phenolic acids and flavonoids other than

anthocyanins are the most probable bioactive fractions of this fruit.

Food supplementation and fortification are considered as most successful and viable

strategies to prevent malnutrition and its consequential health disorders in economically deprived

undernourished population groups of underdeveloped nations. Millions of tons of food waste is

produced annually and is exposed to environment as pollutant garbage thereby threatening bio-

safety of the ecosystem. Meticulous assessment of nutritional and functional features of such

172

bulks and mechanistic exploitation of their food features in various forms is highly appreciated

as environmental friendly food production approach.

Despite the availability of abundant literature to highlight ethnopharmacological and

nutraceutical features of pomegranate and its peel fractions, there have been fewer reports on the

possible toxicological effects, dietary uses, dietary levels and consumption patterns of PoP,

derived extracts and the meal fraction. The present research is primarily an attempt to explore

nutritional, nutraceutical and food stabilization features of PoP and the derived fractions in baked

products i.e. cookies. With a view point to sagaciously exploit these valuable ingredients in

certain food preparations, PoP, PoPx and PM were partially incorporated in cookies to offer

nutritionally enriched and functionally potent food. Limited food use of PoP and PoPx as

ingredients of choice in food systems are either attributed to their astringency or to some extent

suspected anti-nutritional factors. Data pertaining to organoleptic evaluation of the cookies

prepared with increased levels of PoP exhibited PoP supplementation as an acceptable approach

to modulate certain maladies. In order to ensure their biological safety for onward consumption

as food ingredient, PoP, PoPx and the peel bagasse were further evaluated for suspected toxicity

at organoleptically acceptable maximum supplementation levels.

Synthetic antioxidants and antimicrobials, being injudiciously exploited as food

preservatives involve certain health implications therefore replacement of these synthetic food

additives with relatively least to non-toxic natural phytochemicals is referred to be the

sustainable and safe approach in food preservation. Evidently, PoP and its ethnobotanical

extracts have been found to deliver significant antioxidant, antimicrobial and nutraceuticals

properties and are shown to enhance food quality.

Biochemical composition and free radical scavenging features of PoP and PoP

supplemented cookies were evaluated. The study revealed PoP supplementation to significantly

(p<0.05) improve dietary fibers (0.32 – 1.96g/100g), total phenols (90.7 – 161.9mgGAE/100g)

and inorganic residues (0.53 – 0.76g/100g) of cookies. Similarly, owing to its high inorganic

residual levels particularly with the electrolytes, PoP supplementation significantly increased Ca,

K, Fe and Zn levels of supplemented cookies. Almost 50% DPPH radical scavenging activity

was recorded in cookies carrying highest concentration of PoP (7.5%) and phenolic contents.

PoP phenolics of supplemented cookies were shown to reduce oxidative degradation during four

months storage. Present study suggests PoP supplementation as a potential source of

173

microelements and macronutrients particularly carbohydrates and crude fiber in baked products.

Application of PoP in ready to serve foods seems to be a potential disease preventive and

ameliorative approach in tandem with its preservation and nutritional enhancement features.

Exceptionally rich micro-elemental pool, low profiled lipids and higher dietary fibers

contents of pomegranate bulk waste i.e. PoP can be recognized as a tremendous food ingredient

with cardiovascular protective features parallel to its already defined free radicals scavenging

properties. In view of the research findings, present study highlights PoP as an healthier bio

ingredient for food supplementation and suggests further exploration in clinical aspects in terms

of the availability of micro and macro minerals at regular dietary consumption of PoP

supplemented food products. Present study further suggests toxicological safety of the fruit waste

ingredient for any suspected health implication at dose levels followed in the present research

endeavor.

After identifying dose response of PoP on various quality features of supplemented

cookies, PoPx and the meal fraction were supplemented to wheat flour cookies at 0.25 – 1.0%

and 1.5 – 7.5%, respectively. Cookies supplemented with 7.5% PM reduced caloric contents and

significantly (p<0.05) enhanced dietary fiber and inorganic residues of the product. Positive

correlation was observed for cookies phenolics contents with DPPH and FRAP assay. Total

phenolics recovery from PM and PoPx was shown to range from 78.35 to 315.7 mgGAE(100g-1),

respectively. PoPx supplemented cookies, holding significant antioxidant properties, inhibited

thiobarbituric acid number by 67% and reduced growth of aerobic counts (2.04 – 1.30 log10cfu/g)

and yeasts / molds (1.70 – 1.05 log10cfu/g). Sensory evaluation of PM supplemented cookies

indicated relatively lower score for texture and crispiness whilst other treatment combinations

were ranked acceptable at 9 point hedonic scale for all sensory parameters.

PoP being a by-product of fruit processing industry, ranks among the most explored

commodities for its biological, pharmacological and nutritional features. The upshots of the

research on PoP, PoPx and PM highlighted the food features of these potentially viable food

supplements to act not only as a defense against certain critically physiological threats but also

have the potential to serve the purpose of food security and waste management.

Toxicity of PoPx was preliminary evaluated in Artemia salina and ~1000ppm

concentration of the methanolic, ethanolic, acetone and water extracts were found non-lethal in

174

brine shrimps larvae. Toxicological and drug development studies most often implicate rats and

mice model for physiological evaluation and among these, male rats are preferred for their higher

capability to metabolize xenobiotic. Present research confirms PoP and PM @ dose level of 6 %

and PoPx @ 1.0% as non –lethal in rats.PM and PoPx supplementation in cookies at maximum

dose level of 6 % elicit a little or no effect on feed consumption of experimental rats when

compared with placebo suggesting PM and PoPx supplementation to be efficacious and effective

strategy for their use as food ingredients. Similarly, weight gain, protein intake, and protein

digestibility among all the tested groups of experimental rats were found to show little variance

as a function of supplementation of PM and PoPx in cookies. Feed conversion efficiency, being

the function of feed intake and weight gain was however found to decline by~ 9% at 2 – 4% and

0.25 – 0.50% supplementation of PM and PoPx, respectively whereas, the supplementation at

higher doses of PM and PoPx were found to offer quite stable effect on feed conversion

efficiency among treated rats.

Effect of supplementation of PM and PoPx in cookies on organ weight was evaluated and

a slight variation in organ weight was noticed. This difference in organs weight might be

ascribed to lower feed intake response and better tendency of the plant material to prevent fatty

tissues development. Pomegranate peel and all its tested fractions did not present any toxic

response on organ weight and a gradual development in different tissue organs was observed

with progression of body weight. A slight reduction in heart weight was noticed in rats groups

supplied diets supplemented with PoP, PoPx and PM without any drastic disease symptom.

Effect of PoP, PoPx and PM supplementation on serum chemistry of wistar albino rats revealed a

modulatory effect on serum lipids, enzymes and proteins profile however; the anticipated effect

did not breach normal limits of the said parameters thereby indicating all tested fractions to be

safer when dispensed even to highest tested doses i.e. 6% PM and PoPx and 1% PoPx. Serum

triglycerides, total cholesterol, LDL, creatinine and bilirubin contents and enzymes including

ALP, ALT and AST were tested among rats against PoP, PoPx and PM supplementation in

cookies. The results depicted slight but significant reduction in these parameters thereby

suggesting PoP and PoPx supplementation to be a viable approach and these ingredients to be

exploited as mediatory agent in kidney and liver diseases as well as to address

hypercholesterolemia and associated health disorders. Current study further validates PoP and

PoPx to be effective in slightly reducing serum albumin and total protein contents as compared

175

to the control whereas PM at its higher supplementation was witnessed to add protein value in

serum without showing any symptom of toxicity or infection in the tested animal thus declaring

the tested fraction of pomegranate peel as non- toxic in biological subjects at sub-chronic

exposure.

Safety assessment of PoP, PoPx and PM as noticed from hematological parameters of

rats in control and treated rats uncovered no detrimental effect of PoP, PM and PoPx

supplementation. When compared with the control group, rats supplied supplemented diets

presented a significant effect of diet on various hematological parameters including blood cell

counts and cell volume. However; the findings on various parameters including white blood

cells, red blood cells, hemoglobin, platelets, lymphocytes, neutrophils, hematocrits, mean

corpuscular volume and mean corpuscular hemoglobin concentration were quite in range

prescribed for healthy normal rats of the same gender and age group as has been studied in

current research. The study further delineates no abnormal rise or fall in any of the tested

hematological parameter in all tested groups thereby suggesting a non – toxic response of

pomegranate peel and tested fractions at dose levels followed in the present study.

176

RECOMMENDATIONS

PoP phenolics hydro-alcoholic extraction at 25ºC with alcohol – water ratio of 70:30 and

0.2mm particle mesh size improve extracts yield, phenolics recovery and associated

antioxidant activity.

PoPx of Alipuri cultivar possesses tremendous urease inhibitory properties and hence

may be explored as ethnopharmacological drug in treating infections.

PoP of white rind cultivars can be supplemented @6.0% in cookies and similar baked

products without any negative sensorial attribute whereas supplementation of PoPx and

PM can be used up to 1.0% and 7.5%, respectively.

PoPx inhibits lipid oxidation and microbial proliferation in baked goods and may be

substituted with the synthetic group of antioxidants and antimicrobial.

Sub-chronic to chronic exposure of PoP and PoPx as feed ingredient positively modulates

lipid profile and enzymes balance and hence may be implicated for the treatment of

hypercholesterolemia, liver and kidney disorders.

177

REFERENCES

Abdel-Rahim, E.A., El-Beltagi, H.S., Romela, R.M., 2013. White Bean seeds and Pomegranate

peel and fruit seeds as hypercholesterolemic and hypolipidemic agents in albino

rats. Grasas y Aceites 64, 50 – 58.

Abdollahzadeh, S. H., Mashouf, R. Y., Mortazavi, H., Moghaddam, M. H., Roozbahani, N.,

Vahedi, M., 2011. Antibacterial and antifungal activities of Punica granatum peel

extracts against oral pathogens. Journal of Dentistry(Tehran), 8, 1-6.

Adams, L.S., Zhang, Y., Seeram, N.P., Heber, D., Chen, S., 2010. Pomegranate ellagitannin–

derived compounds exhibit antiproliferative and antiaromatase activity in breast

cancer cells in vitro. Cancer Prevention Research 3,108-113.

Adegoke, G.O., Vijay Kumar, M., Gopal Krishna, A.G., Varadaraj, M.C., Sambaiah, K., Lokesh,

B.R., 1998. Antioxidants and lipid oxidation in foods: A critical appraisal. Journal

Food Science and Technology 35, 283–298.

Adiga, S., Tomar, P., Rajput, R.R., 2010. Effect of Punica granatum peel aqueous extract on

normal and dexamethasone suppressed wound healing in wistar rats. International

Journal of Pharmaceutical Sciences Review and Research 5, 134-140.

Afaq, F., Saleem, M., Krueger, C.G., Reed, J.D., Mukhtar, H., 2005. Anthocyanin- and

hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-

kappaB pathways and inhibits skin tumorigenesis in CD-1 mice. International

Journal of Cancer 113, 423-433.

Afaq, F., Zaid, MA., Khan, N., Dreher, M., Mukhtar, H., 2009. Protective effect of pomegranate-

derived products on UVB-mediated damage in human reconstituted skin.

Experimental Dermatology 18, 553-561.

Aguilar, C. N., Aguilera-Carbo, A., Robledo, A., Ventura, J., Belmares, R., Martinez, D.,

Rodríguez-Herrera, R., Contreras, J., 2008. Production of antioxidant

nutraceuticals by solid-state cultures of pomegranate (Punica granatum) peel and

creosote bush (Larrea tridentata) leaves. Food Technology and Biotechnology,

46, 218-222.

178

Aguilera-Carbo, A., Augur, C., Prado-Barragan, L. A., Favela-Torres, E., Aguilar, C. N., 2008.

Microbial production of ellagic acid and biodegradation of ellagitannins. Applied

Microbiology and Biotechnology, 78, 189-199.

Aiyer,H.S., Bouker, K.B., Cook, K.L., Facey, C.O.B., Hu, R., Schwartz, J.L., Shajahan, A.N.,

Hilakivi-Clarke, L., Clarke, R., 2012. Interaction of dietary polyphenols with

molecular signaling pathways of antiestrogen resistance: possible role in breast

cancer recurrence. Hormone Molecular Biology and Clinical Investigation 9,

127–141.

Ajaikumar, K.B., Asheef, M., Babu, B.H., Padikkala, J., 2005. The inhibition of gastric mucosal

injury by Punica granatum L. (pomegranate) methanolic extract. Journal of

Ethnopharmacology 96, 171-176.

Akbarpour, V., Hemmati, K., Sharifani, M., 2009. Physical and chemical properties of

pomegranate (Punica granatum L.) fruit in maturation stage. American-Eurasian

Journal of Agricultural & Environmental Sciences 6, 411-416.

Akhtar S, Ismail T, Riaz M., 2013b. Flaxseed - Miraculous defense against some critical

maladies. Pakistan Journal of Pharmaceutical Sciences 26, 199 – 208.

Akhtar, S., 2015. Food safety challenges—a Pakistan's perspective. Critical Reviews in Food

Science and Nutrition, 55, 219-226.

Akhtar, S., Anjum, F. M., Rehman, S. U., Sheikh, M. A., Farzana, K., 2008. Effect of

fortification on physico-chemical and microbiological stability of whole wheat

flour. Food Chemistry 110, 113-119.

Akhtar, S., Ismail, T., Atukorala, S., Arlappa, N., 2013a. Micronutrients deficiencies in South

Asia – Current status and strategies. Trends in Food Science & Technology 31,55

– 62.

Akhtar, S., Ismail, T., Fraternale, D., Sestili, P., 2015. Pomegranate peel and peel extracts:

Chemistry and food features. Food Chemistry 174, 417-425.

Akhtar, S., Ismail, T., Riaz, M. 2013a. Flaxseed-a miraculous defense against some critical

maladies. Pakistan Journal of Pharmaceutical Sciences26, 199-208.

179

Akhtar, S., Sarker, M. R., Hossain, A. 2014. Microbiological food safety: a dilemma of

developing societies. Critical Reviews in Microbiology, 40, 348-359.

Akpinar-Bayizit, A., Yilmaz-Ersan, L., Ozcan, T., 2012. The therapeutic potential of

pomegranate and its products for prevention of cancer. INTECH Open Access

Publisher.

Al‐Gubory, K.H., Blachier, F., Faure, P., Garrel, C., 2015. Pomegranate peel extract decreases

small intestine lipid peroxidation by enhancing activities of major antioxidant

enzymes. Journal of the Science of Food and Agriculture.

DOI: 10.1002/jsfa.7529.

Al-Jarallah, A., Igdoura, F., Zhang, Y., Tenedero, C. B., White, E. J., MacDonald, M. E.,

Trigatti, B. L., 2013. The effect of pomegranate extract on coronary artery

atherosclerosis in SR-BI/APOE double knockout mice. Atherosclerosis 228, 80-

89

Al-Maiman, S.A., and Ahmad, D., 2002. Changes in physical and chemical properties during

pomegranate (Punica granatumL.) fruit maturation. Food Chemistry 76, 437–441.

Al-said, F., Opara, L., Al-yahyai, R. 2009. Physico-chemical and textural quality attributes of

pomegranate cultivars (Punica granatum L.) grown in the Sultanate of Oman.

Journal of Food Engineering90, 129-134.

Al-Sayed, H.M.A., Ahmed, A.R., 2013. Utilization of watermelon rinds and sharlyn melon peels

as a natural source of dietary fiber and antioxidant in cake. Annals of Agricultural

Sciences 58, 83 – 95.

Altunkaya A., Hedegaard R.V., Brimer L., Gokmen V., Skibsted L.H., 2013a. Antioxidant

capacity versus chemical safety of wheat bread enriched with pomegranate peel

powder. Food & Function 4, 722 – 727.

Altunkaya, A., Hedegaard, R.V., Harholt, J., Brimer, L., Gokmen, V., Skibsted, L.H., 2013b.

Palatability and chemical safety of apple juice fortified with pomegranate peel

extract. Food & Function 4, 1468 – 1473.

Al-Zoreky, N. S. 2009. Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels.

International Journal of Food Microbiology 134, 244-248.

180

Al-Zoreky, N., Nakahara, K., 2001. Antioxidant activity of some edible Yemeni plants evaluated

by Ferrylmyoglobin/ABTS·+ Assay. Food Science & Technology Research7,

141-144.

Al-Zoreky, N., Nakahara, K., 2003. Antibacterial activity of extracts from some edible plants

commonly consumed in Asia. International Journal of Food Microbiology80,

223–230.

Amrutesh, S., 2011. Dentistry & ayurveda V- An evidence based approach. International Journal

of Clinical Dental Science 2, 3-9.

Amyrgialaki, E., Makris, D.P., Mauromoustakos, A., Kefalas, P., 2014. Optimisation of the

extraction of pomegranate (Punica granatum) husk phenolics using water/ethanol

solvent systems and response surface methodology. Industrial Crops and

Products 59, 216-222.

Anderson, J.W., Baird, P., Davis, Jr R.H., Ferreri, S., Knudtson, M., Koraym, A., Waters, V.,

Williams, C.L., 2009. Health benefits of dietary fiber. Nutrition Reviews 67, 188

– 205.

Anoosh, E., Mojtaba, E., Fatemeh, S., 2012. Antioxidant activity of juice and peel extract of

three variety of Pomegranate and the effect of pomegranate juice on the plasma

lipids. International Journal of Biosciences 2, 116-123.

AOCS. 1993. Official methods of the American oil Chemists Society (4th ed.). Champaign, IL:

American Oil Chemists Society.

APHA. 1992.Compendium of methods for the microbiological examination of foods. 3rd Ed.,

American Public Health Association, Washington, D.C.

Arapitsas, P., 2012.Hydrolyzable tannin analysis in food. Food Chemistry 135, 1708–1717.

Ardekani, Shams, M.R., Hajimahmoodi, M., Oveisi, M.R., Sadeghi, N., Jannat, B., Ranjbar,

A.M., Gholam, N., Moridi, T., 2011. Comparative antioxidant activity and total

flavonoid content of Persian pomegranate (Punica granatum L.) cultivars. Iran.

Journal of Pharmacy Research 10, 519.

Arora, A., Nair, M. G., Strasburg, G. M., 1998. Structure–activity relationships for antioxidant

activities of a series of flavonoids in a liposomal system. Free Radical Biology

and Medicine, 24, 1355-1363.

181

Arun, N., Singh, D.P., 2012. Punica granatum: a review on pharmacological and therapeutic

properties. International Journal of Pharmaceutical Sciences and Research 3,

1240-1245.

Asgary, S., Keshvari, M., Sahebkar, A., Hashemi, M., Rafieian-Kopaei, M., 2013. Clinical

investigation of the acute effects of pomegranate juice on blood pressure and

endothelial function in hypertensive individuals. ARYA Atherosclerosis 9, 326-

331.

Asghari, G., Sheikholeslami, S., Mirmiran, P., Chary, A., Hedayati, M., Shafiee, A., Azizi, F.

2012. Effect of pomegranate seed oil on serum TNF-α level in dyslipidemic

patients. International Journal of Food Sciences and Nutrition 63, 368-371.

Ashoush, I. S., El-Batawy, O. I., El-Shourbagy, G. A., 2013. Antioxidant activity and

hepatoprotective effect of pomegranate peel and whey powders in rats. Annals of

Agricultural Sciences 58, 27-32.

Assmann, G., Nofer, J. R., 2003. Atheroprotective effects of high-density lipoproteins. Annual

Review of Medicine 54, 321-341.

Atta-UR-Rehman, Choudhary, M.I., Thomsen, W., 2001. Bioassay Techniques for Drug

Development. Harwood Academic Publisher, Netherlands.

Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R., Presser, D.,

Fuhrman, B., 2000. Pomegranate juice consumption reduces oxidative stress,

atherogenic modifications to LDL, and platelet aggregation: studies in humans

and in atherosclerotic apolipoprotein E–deficient mice. The American Journal of

Clinical Nutrition 71, 1062-1076.

Aviram, M., Volkova, N., Coleman, R., Dreher, M., Reddy, M.K., Ferreira, D., Rosenblat, M.,

2008. Pomegranate phenolics from the peel, arils and flowers are antiatherogenic:

studies in vivo in atherosclerotic apolipoprotein e-deficient (E0) mice and in vitro

in cultured macrophages and lipoproteins. Journal of Agricultural and Food

Chemistry 56, 1148–1157.

Babior, B.M.., 2000. Phagocytes and oxidative stress. The American Journal of Medicine 109,

33-44.

182

Bachoual, R., Talmoudi, W., Boussetta, T., Braut, F., El-Benna, J., 2011. An aqueous

pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and

attenuates lung inflammation in mice. Food and Chemical Toxicology 49, 1224-

1228.

Banerjee, S., Kambhampati, S., Haque, I., Banerjee, S.K., 2011. Pomegranate sensitizes

Tamoxifen action in ER-α positive breast cancer cells. Journal of Cell

Communication and Signaling 5, 317-324.

Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of

antioxidant power: The FRAP assay. Analytical Biochemistry 239, 70–76.

Bertagnolli, S.M.M., Silveira, M.L.R., Fogaça, A.D.O., Umann, L., Penna, N.G., 2014. Bioactive

compounds and acceptance of cookies made with Guava peel flour. Food Science

and Technology (Campinas) 34, 303-308.

Bhandary, B. S. K., Sharmila, K. P., Kumari, N. S., Bhat, S. V., 2013. Acute and subacute

toxicity study of the ethanol extracts of Punica granatum (Linn). Whole fruit and

seeds and synthetic ellagic acid in swiss albino mice. Asian Journal of

Pharmaceutical and Clinical Research, 6, 192-198.

Bialonska, D., Kasimsetty, S.H., Schrader, K.K., Ferreira, D., 2009.The effect of pomegranate

(Punica granatum L.) byproducts and ellagitannins on the growth of human gut

bacteria. Journal of Agricultural and Food Chemistry 57, 8344–8349.

Billett, H. H. (1990). Hemoglobin and hematocrit. In: Walker, H.K., Hall, W.D., Hurst, J.W.,

(eds.), Clinical methods: The history, physical, and laboratory examination.

Boston: Butterworths.

Bom, D.C., Gray, K., Potineni, R.V., Ommeren, E.V., 2012. Aftertaste masking. US Patents.

Patent no. US 20120244271A1.

Braga, L.C., Shupp, J.W., Cummings, C., Jett, M., Takahashi, J.A., Carmo, L.S., Chartone-

Souza, E., Nascimento, A.M., 2005. Pomegranate extract inhibits Staphylococcus

aureus growth and subsequent enterotoxin production. Journal of


183

Burda, S., Oleszek, W., 2001. Antioxidant and antiradical activities of flavonoids. Journal of

Agricultural and Food Chemistry, 49, 2774-2779.

Cam, M., Hısıl, Y., 2010. Pressurised water extraction of polyphenols from pomegranate peels.

Food Chemistry123, 878–885.

Carballo, L.J., Hernandez-Inda, L.Z., Perez, P., Gravalos, M.D., 2002. A comparison between

two brine shrimp assays to detect in vitro cytotoxicity in marine natural products.

BMC Biotechnology 2, 1-10.

Celik, I., Temur, A., Isik, I., 2009. Hepatoprotective role and antioxidant capacity of

pomegranate (Punica granatum) flowers infusion against trichloroacetic acid-

exposed in rats. Food and chemical toxicology : an international journal published

for the British Industrial Biological Research Association 47, 145-149.

Cerda, B., Ceron, J. J., Tomas-Barberan, F. A., Espin, J. C., 2003b. Repeated oral administration

of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is

not toxic. Journal of Agricultural and Food Chemistry 51, 3493–3501.

Cerda, B., Espın, J.C., Parra, A., Martınez, P., Tomas-Barberan, F.A., 2004. The potent in vitro

antioxidant ellagitannins from pomegranate juice are metabolized into

bioavailable but poor antioxidant hydroxy- 6H-dibenzopyran-6-one derivatives by

the colonic microflora of healthy humans. European Journal of Nutrition 43, 205–

220.

Cerda, B., Llorach, R., Ceron, J.J., Espin, J.C., and Tomas-Barberan, F.A., 2003a. Evaluation of

the bioavailability and metabolism in the rat of punicalagin, an antioxidant

polyphenols from pomegranate juice. European Journal of Nutrition 42, 18–28.

Charles River Laboratories. 1998. Baseline hematology and clinical chemistry values for Charles

River Rats – (CRL: (WI) BR) as a function of sex and age. Wilmington, MA.

Chemat, F., Grondin, I., Costes, P., Moutoussamy, L., Sing, A.S.C., Smadja, J., 2004. High

power ultrasound effects on lipid oxidation of refined sunflower oil. Ultrasonics

Sonochemistry 11, 281-285.

Chen, Z., Bertin, R., Froldi, G., 2013. EC50 estimation of antioxidant activity in DPPH assay

using several statistical programs. Food Chemistry 138, 414-420.

184

Chevion, M., 1988. A site-specific mechanism for free radical induced biological damage: The

essential role of redox-active transition metals. Free Radical Biology and

Medicine 5, 27-37.

Chidambara Murthy, K.N., Jayaprakasha, G.K., Singh, R.P., 2002. Studies on antioxidant

activity of pomegranate (Punica granatum) peel extracts. Journal of Food and

Agricultural Chemistry 50, 4791 – 4795.

Choi, J.G., Kang, O.H., Lee, Y.S., Chae, H.S., Oh, Y.C., Brice, O.O., Kwon, D.Y.,2011. In vitro

and in vivo antibacterial activity of Punica granatum peel ethanol extract against

Salmonella. Evidence-Based Complimentary and Alternate Medicine1–

8.doi:10.1093/ecam/nep105.

Collins, M.D., Gibson, G.R., 1999. Probiotics, prebiotics and symbiotic: approaches for the

nutritional modulation of microbial ecology. American Journal of Clinical

Nutrition69, 1052–1057.

Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12,

564-582.

Dacie, J. V., Lewis, S. M., 1984. Estimation of plasma haemoglobin. Practical Haematology 6th

Ed., Churchill Livingstone, London, 139-40.

Daglioglu, O., Tasan, M., Gecgel, U., Daglioglu, F., 2004. Changes in oxidative stability of

different bakery products during shelf life. Food Science and Technology

Research 10, 464 – 468.

De Castro, E. S., Boyd, E. M., 1968. Organ weights and water content of rats fed on protein-

deficient diets. Bulletin of the World Health Organization 38, 971.

de Nigris, F., Williams-Ignarro, S., Lerman, L.O., Crimi, E., Botti, C., Mansueto, G.,

D'Armiento, F.P., De Rosa, G., Sica, V., Ignarro, L.J., Napoli, C., 2005.

Beneficial effects of pomegranate juice on oxidation-sensitive genes and

endothelial nitric oxide synthase activity at sites of perturbed shear stress.

Proceedings of the National Academy of Sciences USA 102(13), 4896-4901.

de Oliveira, E. P., Burini, R. C., 2012. High plasma uric acid concentration: causes and

consequences. Diabetology and Metabolic Syndrome 4:12.

185

Dell'Agli, M., Galli, G.V., Bulgari, M., Basilico, N., Romeo, S., Bhattacharya, D., Taramelli, D.,

Bosisio, E., 2010. Ellagitannins of the fruit rind of pomegranate (Punica

granatum) antagonize in vitro the host inflammatory response mechanisms

involved in the onset of malaria. Malaria Journal 9, 208.

Devatkal, S. K., Thorat, P. R., Manjunatha, M., Anurag, R. K., 2012. Comparative antioxidant

effect of aqueous extracts of curry leaves, fenugreek leaves and butylated

hydroxytoluene in raw chicken patties. Journal of Food Science and

Technology, 49, 781-785.

Devatkal, S. K., Thorat, P., and Manjunatha, M., 2014. Effect of vacuum packaging and

pomegranate peel extract on quality aspects of ground goat meat and nuggets.

Journal of Food Science and Technology 51, 2685-2691.

Dhingra D, Michael M, Rajput H, Patil RT. (2012). Dietary fibers in foods: a review. Journal of

Food Science and Technology 49, 255 – 266.

Di Silvestro, R.A., Di Silvestro, D.J., Di Silvestro, D.J., 2009. Pomegranate extract mouthrinsing

effects on saliva measures relevant to gingivitis risk. Phytotherapy Research

23(8), 1123-1127.

Drewnowski, A., Gomez-Carneros, C., 2000. Bitter taste, phytonutrients, and the consumer: a

review. American Journal of Clinical Nutrition 72, 1424-1435.

Duman, A.D., Ozgen, M., Dayisoylu, K.S., Erbil, N., Durgac, C., 2009. Antimicrobial activity of

six pomegranate (Punica granatum L.) varieties and their relation to some of their

pomological and phytonutrient characteristics. Molecules 14, 1808-1817.

El Kar, C., Ferchichi, A., Attia, F., Bouajila, J., 2011. Pomegranate (Punica granatum) juices:

chemical composition, micronutrient cations, and antioxidant capacity. Journal of

Food Science 76, C795-C800.

El-Gharably, A.M.A., Ashoush, I.S., 2011.Utilization impact of adding pomegranate rind powder

and red beet powder as natural antioxidant on quality characteristics of beef

sausages. World Journal of Dairy and Food Science 6, 86–97.

http://link.springer.com/search?facet-author=%22Suresh+K.+Devatkal%22

http://link.springer.com/search?facet-author=%22Pramod+Thorat%22

http://link.springer.com/search?facet-author=%22M.+Manjunatha%22

186

El-Sherbini, G. M., Ibrahim, K. M., El Sherbiny, E. T., Abdel-Hady, N. M., Morsy, T. A., 2010).

Efficacy of Punica granatum extract on in-vitro and in-vivo control of

Trichomonas vaginalis. Journal of the Egyptian Society of Parasitology 40, 229-

244.

FAO/WHO/UNU. 2007. Expert consultation on protein and amino acid requirements in human

nutrition. WHO Technical Report Series No.935.

Faria, A., Monteiro, R., Mateus, N., Azevedo, I., Calhau, C., 2007. Effect of pomegranate

(Punica granatum) juice intake on hepatic oxidative stress. European Journal of

Nutrition 46, 271-278.

Fawole, O.A., Makunga, N.P., Opara, U.L., 2012. Antibacterial, antioxidant and tyrosinase-

inhibition activities of pomegranate fruit peel methanolic extract. BMC

Complimentary and Alternate Medicine 12, 200.

Fawole, O.A., Opara, U.L., 2012. Composition of trace and major minerals in different parts of

pomegranate (Punica granatum) fruit cultivars. British Food Journal 114, 1518 –

1532.

Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., Parkin, D.M., 2010. GLOBOCAN

2008: cancer incidence and mortality worldwide. Lyon: International Agency for

Research on Cancer.

Fischer, U. A., Jaksch, A. V., Carle, R., Kammerer, D. R., 2013. Influence of origin source,

different fruit tissue and juice extraction methods on anthocyanin, phenolic acid,

hydrolysable tannin and isolariciresinol contents of pomegranate (Punica

granatum L.) fruits and juices. European Food Research and Technology, 237,

209-221.

Fischer, U.A., Carle, R., Kammerer, D.R., 2011. Identification and quantification of phenolic

compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and

differently produced juices by HPLC-DAD–ESI/MS. Food Chemistry 127, 807-

821.

187

Ford, E. S., Mokdad, A. H., Giles, W. H., Mensah, G. A., 2003. Serum total cholesterol

concentrations and awareness, treatment, and control of hypercholesterolemia

among US adults findings from the National Health and Nutrition Examination

Survey, 1999 to 2000. Circulation 107, 2185-2189

Forgacs, I., Loganayagam, A., 2008. Overprescribing proton pump inhibitors. BMJ 336, 2–3.

Fowler, Z.L., Baron, C.M., Panepinto, J.C., Koffas, M.A., 2011. Melanization of flavonoids by

fungal and bacterial laccases. Yeast 28, 181–188.

Frost and Sullivan. 2013. Natural shelf-life extension food additives set to overtake synthetic

antioxidants. North - American shelf life extension food additives market.

Retrieved from: http://en.prnasia.com/story/73614-0.shtml.Accessed on: 18-01-

2013.

Gibson, G.R., Probert, H.M., Van Loo, J., Rastall, R.A., Roberfroid, M.B., 2004. Dietary

modulation of the colonic microbiota: updating the concept of prebiotics.

Nutrition Research Reviews17, 259–275.

Gil, M.I., Tomas-Barberan, F.A., Hess-Pierce, B., Holcroft, D.M., Kader, A.A., 2000.

Antioxidant activity of pomegranate juice and its relationship with phenolic

composition and processing. Journal of Agricultural and Food Chemistry 48,

4581-4589.

Gopalakrishnan, S., Benny, P. J., 2009. In vitro antimicrobial properties of Punica granatum

extract on bacteria causing urinary tract infections. Indian Drugs, 46, 9.

Gullon, B., Pintado, M. E., Pérez-Álvarez, J. A., Viuda-Martos, M., 2016. Assessment of

polyphenolic profile and antibacterial activity of pomegranate peel (Punica

granatum) flour obtained from co-product of juice extraction. Food Control 59,

94-98.

Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Jiang, Y., 2003. Antioxidant activities of peel, pulp, and

seed fractions of common fruits as determined by FRAP assay. Nutrition

Research 23, 1719-1726.

http://en.prnasia.com/story/73614-0.shtml

188

Haidari, M., Ali, M., Casscells, S. W., Madjid, M., 2009. Pomegranate (Punica granatum)

purified polyphenol extract inhibits influenza virus and has a synergistic effect

with oseltamivir. Phytomedicine 16, 1127-1136.

Hajimahmoodi, M., Shams-Ardakani, M., Saniee, P., Siavoshi, F., Mehrabani, M., Hosseinzadeh,

H., Foroumadi, P., Safavi, M., Khanavi, M., Akbarzadeh, T.,& Shafiee, A. 2011.

In vitro antibacterial activity of some Iranian medicinal plant extracts against

Helicobacter pylori. Natural Product Research 25, 1059-1066

Han, H.M., Koh, B.K., 2011. Antioxidant activity of hard wheat flour, dough and bread prepared

using various processes with the addition of different phenolic acids. Journal of

the Science of Food and Agriculture 91, 604 – 608.

Han, J., Weng, X., Bi, K., 2008. Antioxidants from a Chinese medicinal herb – Lithospermum

erythrorhizon. Food Chemistry106, 2–10.

Harikrishnan, R., Kim, J. S., Kim, M. C., Balasundaram, C., Heo, M. S., 2012. Pomegranate

enriched diet enhances the hematology, innate immune response, and disease

resistance in olive flounder against Philasterides dicentrarchi. Veterinary

parasitology 187, 147-156

Haslam, E., 1996. Natural polyphenols (vegetable tannins) as drugs: possible modes of

action. Journal of Natural Products, 59, 205-215.

Hassanpour Fard, M., Ghule, A.E., Bodhankar, S.L., Dikshit, M., 2011. Cardioprotective effect

of whole fruit extract of pomegranate on doxorubicin-induced toxicity in rat.

Pharmaceutical Biology 49, 377-382.

Hayrapetyan, H., Hazeleger, W.C., Beumer, R.R., 2012. Inhibition of Listeria monocytogenes by

pomegranate (Punica granatum) peel extract in meat pate at different

temperatures. Food Control 23,66–72.

Heber, D., 2011. Pomegranate ellagitannins. In: Herbal medicine: biomolecular and clinical

aspects. Benzie, I.F.F. and Wachtel-Galor, S., Eds., 2nd ed. CRC Press, Boca

Raton (FL).

Heber, D., Seeram, N.P., Wyatt, H., Henning, S.M., Zhang, Y., Ogden, L.G., Dreher, M., Hill,

J.O., 2007. Safety and antioxidant activity of a pomegranate ellagitannin-enriched

189

polyphenol dietary supplement in overweight individuals with increased waist

size. Journal of Agricultural and Food Chemistry55, 10050–10054.

Heim, K. E., Tagliaferro, A. R., Bobilya, D. J., 2002. Flavonoid antioxidants: chemistry,

metabolism and structure-activity relationships. The Journal of Nutritional

Biochemistry 13, 572-584.

Hirano, R., Sasamoto, W., Matsumoto, A., Itakura, H., Igarashi, O., Kondo, K., 2001.

Antioxidant ability of various flavonoids against DPPH radicals and LDL

oxidation. Journal of Nutritional Science and Vitaminology47, 357-362.

Hollman, P.C.H., Arts, I.C.W., 2000. Flavonols, flavones and flavanols-nature, occurrence and

dietary burden. Journal of the Science of Food and Agriculture80, 1081–1093.

Holloszy, J. O., Oscai, L. B., Don, I. J., Mole, P. A., 1970. Mitochondrial citric acid cycle and

related enzymes: adaptive response to exercise. Biochemical and Biophysical

Research Communications 40, 1368-1373.

Hossin, F.L.A., 2009. Effect of Pomegranate (Punica granatum) peels and it’s extract on obese

hypercholesterolemic rats. Pakistan Journal of Nutrition 8, 1251-1257.

Houston, M., 2012. The role of nutraceutical supplements in the treatment of dyslipidemia. The

Journal of Clinical Hypertension 14, 121-132.

Howell, A. B., D'Souza, D. H., 2013. The pomegranate: effects on bacteria and viruses that

influence human health. Evidence-Based Complementary and Alternative

Medicine, 2013.

Hu, M., 2007. Commentary: bioavailability of flavonoids and polyphenols: Call to arms.

Molecular Pharmaceutics4, 803–806.

Iqbal, S., Haleem, S., Akhtar, M., Zia-ul-Haq, M., Akbar, J., 2008. Efficiency of pomegranate

peel extracts in stabilization of sunflower oil under accelerated conditions. Food

Research International 41, 194–200.

Ismail, T., Akhtar, S., Riaz, M., Ismail, A., 2014. Effect of pomegranate peel supplementation on

nutritional, organoleptic and stability properties of cookies. International Journal

Food Science Nutrition 65, 661-666.

190

Ismail, T., Sesteli, P., Akhtar, S., 2012. Pomegranate peel and fruit extracts: a review of potential

anti-inflammatory and anti-infective effects. Journal of Ethnopharmacology 143,

397–405.

Jaillon, S., Galdiero, M. R., Del Prete, D., Cassatella, M. A., Garlanda, C., Mantovani, A., 2013.

Neutrophils in innate and adaptive immunity. In Seminars in Immunopathology,

35(4), 377-394. Springer-Verlag.

Jeltema, M.A., Zabik, M.E., Thiel, L.J., 1983. Prediction of cookie quality from dietary fiber

components. Cereal Chemistry 60, 227–230.

Jeune, M. L., Kumi-Diaka, J., Brown, J., 2005. Anticancer activities of pomegranate extracts and

genistein in human breast cancer cells. Journal of Medicinal Food 8, 469-475.

Johanningsmeier, S.D., Harris, G.K., 2010. Pomegranate as a functional food and nutraceutical

source. Annual Reviews in Food Science and Technology 2, 181–201.

Joseph, M. M., Aravind, S. R., George, S. K., Varghese, S., Sreelekha, T. T., 2013. A

galactomannan polysaccharide from Punica granatum imparts in vitro and in vivo

anticancer activity. Carbohydrate Polymers 98, 1466-1475.

Jung, H. J. G., Fahey, G. C., Merchen, N. R., 1983. Effects of ruminant digestion and

metabolism on phenolic monomers of forages. British Journal of Nutrition 50,

637-651.

Kanatt, S.R., Chander, R., Sharma, A., 2010. Antioxidant and antimicrobial activity of

pomegranate peel extract improve shelf life of chicken products. International

Journal of Food Science and Technology 45, 216–222.

Kanavos, P., 2006. The rising burden of cancer in the developing world. Annals of oncology :

official journal of the European Society for Medical Oncology / ESMO 17 Suppl

8, viii15-viii23.

Kaplan, M., Hayek, T., Raz, A., Coleman, R., Dornfeld, L., Vaya, J., Aviram. M., 2001.

Pomegranate juice supplementation to atherosclerotic mice reduces macrophage

lipid peroxidation, cellular cholesterol accumulation and development of

atherosclerosis. Journal of Nutrition 131, 2082–2089.

191

Karaaslan, M., Vardin, H., Varliklioz, S., Yilmaz, F.M., 2014. Antiproliferative and antioxidant

activities of Turkish pomegranate (Punica granatum L.) accessions. International

Journal of Food Science and Technology 49, 82–90.

Karanewsky, D.S., Fotsing, J.R., Tachdjian, C., Arellano, M., 2012. Compounds that inhibit

(Block) bitter taste in composition and use thereof. US Patents. Patent no. US

20120088796 A1.

Kasai, K., Yoshimura, M., Koga, T., Arii, M., Kawasaki, S., 2006. Effects of oral administration

of ellagic acid-rich pomegranate extract on ultraviolet-induced pigmentation in

the human skin. Journal of Nutritional Science and Vitaminology (Tokyo) 52,

383-388.

Kelloff, G.J., Boone, C.W., Crowell, J.A., Steele, V.E., Lubet, R., Sigman, C.C., 1994.

Chemopreventive drug development: perspectives and progress. Cancer

epidemiology, biomarkers & prevention : a publication of the American

Association for Cancer Research, cosponsored by the American Society of

Preventive Oncology 3, 85-98.

Khan, G.N., Gorin, M.A., Rosenthal, D., Pan, Q., Bao, L.W., Wu, Z.F., Newman, R.A., Pawlus,

A.D., Yang, P., Lansky, E.P., Merajver, S.D., 2009. Pomegranate fruit extract

impairs invasion and motility in human breast cancer. Integrative Cancer

Therapies 8, 242-253.

Khateeb, J., Gantman, A., Kreitenberg, A.J., Aviram, M., Fuhrman, B., 2010. Paraoxonase 1

(PON1) expression in hepatocytes is upregulated by pomegranate polyphenols: a

role for PPAR-gamma pathway. Atherosclerosis 208(1), 119-125.

Kim, O. Y., Lee, S. M., Do, H., Moon, J., Lee, K. H., Cha, Y. J., Shin, M. J., 2012. Influence of

Quercetin‐rich Onion peel extracts on adipokine expression in the visceral adipose

tissue of rats. Phytotherapy Research26, 432-437.

Kim, S.B., Kim, I.S., Yeum, D.M., Park, Y.H., 2002. Mutagenicity of maillard reaction products

from D-glucose amino acid mixtures and possible roles of active oxygen in

mutagenicity. Mutation Research 254, 65–69.

http://onlinelibrary.wiley.com/doi/10.1111/ijfs.2013.49.issue-1/issuetoc

192

Kim, W., Flamm, S. L., Di Bisceglie, A. M., Bodenheimer, H. C., 2008. Serum activity of

alanine aminotransferase (ALT) as an indicator of health and disease. Hepatology

47, 1363-1370.

Knight, R.A., Menlove, E.M., 1961. Effect of the bread‐baking process on destruction of certain

mould spores. Journal of Science of Food and Agriculture 12, 653-656.

Koponen, J. M., Happonen, A. M., Mattila, P. H., Törrönen, A. R., 2007. Contents of

anthocyanins and ellagitannins in selected foods consumed in Finland. Journal of

Agricultural and Food Chemistry 55, 1612-1619.

Kotwal, G.J., 2008. Genetic diversity-independent neutralization of pandemic viruses (e.g. HIV),

potentially pandemic (e.g. H5N1 strain of influenza) and carcinogenic (e.g. HBV

and HCV) viruses and possible agents of bioterrorism (variola) by enveloped

virus neutralizing compounds (EVNCs). Vaccine 26, 3055-3058.

Koyama, S., Cobb, L. J., Mehta, H. H., Seeram, N. P., Heber, D., Pantuck, A. J., Cohen, P.,

2010. Pomegranate extract induces apoptosis in human prostate cancer cells by

modulation of the IGF–IGFBP axis. Growth Hormone & IGF Research 20, 55-62.

Kulkarni, A.S., Joshi, D.C., 2013. Effect of replacement of wheat flour with pumpkin powder on

textural and sensory qualities of biscuit. International Food Research Journal 20,

587–591.

Kumari, A., Dora, J., Kumar, A., Kumar, A., 2012. Pomegranate (Punica granatum) – Overview.

International Journal Pharmaceutical and Chemical Sciences 1, 1218–1222.

Land, D.G., Shepherd, R., 1988. Scaling and ranking methods. In: Piggott JR, ed. Sensory

analysis of foods. London: Elsevier Applied Science, 155–185.

Lansky, E., Shubert, S., Neeman, I., 2004. Pharmacological and therapeutic properties of

pomegranate. CIHEAM-Options Mediterraneennes xx, 231-235.

Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for

prevention and treatment of inflammation and cancer. Journal of


193

Larrosa, M., Garcia-Conesa, M.T., Espin, J.C., Tomas-Barberan, F.A., 2010. Ellagitannins,

ellagic acid and vascular health. Molecular Aspects of Medicine 31, 513-539.

Larrosa, M., Gonzalez-Sarrias, A., Garcia-Conesa, M.T., Tomas-Barberan, F.A., Espin, J.C.,

2006. Urolithins, ellagic acid-derived metabolites produced by human colonic

microflora, exhibit estrogenic and antiestrogenic activities. Journal of Agricultural

and Food Chemistry 54, 1611-1620.

Lee, C.J., Chen, L.G., Liang, W.L., Wang, C.C., 2010. Anti-inflammatory effects of Punica

granatum Linne in vitro and in vivo. Food Chemistry 118, 315-322.

Lee, S.I., Kim, B.S., Kim, K.S., Lee, S., Shin, K.S., Lim, J.S., 2008. Immune-suppressive

activity of punicalagin via inhibition of NFAT activation. Biochemical and

Biophysical Research Communications 371, 799-803.

Lester, R., Schmid, R., 1964. Bilirubin metabolism. New England Journal of Medicine 270, 779-

786.

Levrat-Verny, M. A., Coudray, C., Bellanger, J., Lopez, H. W., Demigne, C., Rayssiguier, Y.

and Remesy, C. 1999. Wholewheat flour ensures higher mineral absorption and

bioavailability than white wheat flour in rats. Brazilian Journal of Nutrition 82,

17-21.

Li, G., Xu, Y., Wang, X., Zhang, B., Shi, C., Zhang, W., Xia, X., 2014. Tannin-rich fraction

from pomegranate rind damages membrane of Listeria monocytogenes.

Foodborne Pathogens and Disease 11, 313-319.

Li, Y., Guo, C., Yang, J., Wei, J., Xu, J., Cheng, S., 2006. Evaluation of antioxidant properties of

pomegranate peel extract in comparison with pomegranate pulp extract. Food

Chemistry 96, 254-260.

Lim, C.S.H., Lim, S.L., 2013. Ferric reducing capacity versus ferric reducing antioxidant power

for measuring total antioxidant capacity. Laboratory Medicine 44, 51-55.

Lu, J., Yuan, Q., 2008. A new method for ellagic acid production from pomegranate husk. .

Journal of Food Process Engineering 31, 443-454.

194

Machado, T.B., Pinto, A.V., Pinto, M.C., Leal, I.C., Silva, M.G., Amaral, A.C., Kuster, R.M.,

Netto-dosSantos, K.R., 2003. In vitro activity of Brazilian medicinal plants,

naturally occurring naphthoquinones and their analogues, against methicillin-

resistant Staphylococcus aureus. International Journal of Antimicrobial Agents

21, 279-284.

Maisuthisakul, P., Gordon, M.H., Pongsawatmanit, R., Suttajit, M., 2007. Enhancing the

oxidative stability of rice crackers by addition of the ethanolic extract of

phytochemicals from Cratoxylum formosum Dyer. Asia Pacific Journal of Clinical

Nutrition16, (Suppl 1), 37–42.

Malik, A., Afaq, F., Sarfaraz, S., Adhami, V.M., Syed, D.N., Mukhtar, H., 2005. Pomegranate

fruit juice for chemoprevention and chemotherapy of prostate cancer. Proceedings

of the National Academy of Sciences of the United States of America 102, 14813-

14818.

Malviya, S., Jha, A., Hettiarachchy, N. 2014. Antioxidant and antibacterial potential of

pomegranate peel extracts. Journal of Food Science and Technology51, 4132-

4137.

Manasathien, J., Indrapichate, K., Intarapichet, K., 2012. Antioxidant activity and bioefficacy of

pomegranate Punica granatum Linn. peel and seed extracts. Global Journal of

Pharmacology 6,131-141.

Manasathien, J., Kupittayanant, S., Indrapichate, K., 2011. Protective efficacy of pomegranate

(PunicagranatumLinn.,Punicaceae) peel ethanolic extracts on UVB-irradiated rat

skin. American-Eurasian Journal of Toxicological Sciences 3, 250-258.

Maqsood, S., Benjakul, S., Shahidi, F., 2013. Emerging role of phenolic compounds as natural

food additives in fish and fish products. Critical Reviews in Food Science and

Nutrition 53, 162-179.

Marschner, H., 1995. Mineral nutrition in higher plant. Academic Press, London.

Martin, K.R., Appel, C.L., 2010. Polyphenols as dietary supplements: A double-edged sword.

Journal of Nutrition and Dietary Supplement2, 1–12.

195

McCarrell, E. M., Gould, S. W., Fielder, M. D., Kelly, A. F., El Sankary, W., Naughton, D. P.,

2008. Antimicrobial activities of pomegranate rind extracts: enhancement by

addition of metal salts and vitamin C. BMC Complementary and Alternative

Medicine 8, 64.

McCarron, D.A., Reusser, M.E., 2001. Are low intakes of calcium and potassium important

causes of cardiovascular disease? American Journal of Hypertension 14, 206–212.

McMaster, Y., Tennant, R., Clubb, J. S., Neale, F. C., Posen, S., 1962. Serum alkaline

phosphatase. Gastroenterology 42, 431.

Megyesi, C., Samols, E., Marks, V., 1967. Glucose tolerance and diabetes in chronic liver

disease. The Lancet 290, 1051-1056.

Mehru, N., Rathinam, X., Subramaniam, S., Aiyalu, R., Sreenivasan, S., Reenivasan, S., 2008.

Antimicrobial Activity and Toxicity of Punica granatum L. Peel. Journal of

Applied Biological Sciences 2, 57-9.

Menezes, S.M.S., Cordeiro, L.C., Viana, G.S.B., 2006. Punica granatum (Pomegranate) Extract

Is Active Against Dental Plaque. Journal of Herbal Pharmacotherapy 6, 79-92.

Mildner‐Szkudlarz, S., Bajerska, J., Zawirska‐Wojtasiak, R., Gorecka, D., 2013. White grape

pomace as a source of dietary fibre and polyphenols and its effect on physical and

nutraceutical characteristics of wheat biscuits. Journal of Science of Food and

Agriculture 93, 389-395.

Mirdehghan, S.H., Rahemi, M., 2007. Seasonal changes of mineral nutrients and phenolics in

pomegranate (Punica granatum L.) fruit. Scientia Horticulturae 111,120–127.

Mirdehghan, S.H., Rahemi, M., 2007. Seasonal changes of mineral nutrients and phenolics in

pomegranate (Punica granatum L.) fruit. Scientia Horticulturae 111, 120–127.

Mobley, H.L., Hausinger, R.P., 1989. Microbial ureases: significance, regulation, and molecular

characterization. Microbiology Reviews 53, 85-108.

Mochizuki, T., Hara, S., 2000. Usefulness of the low protein rice on the diet therapy in patients

with chronic renal failure. Nihon Jinzo Gakkaishi 42, 24-29.

Murthy, K.N., Reddy, V.K., Veigas, J.M., Murthy, U.D., 2004. Study on wound healing activity

of Punica granatum peel. Journal of Medicinal Food 7, 256-259.

http://informahealthcare.com/action/doSearch?Contrib=Menezes%2C+S+M+S

http://informahealthcare.com/action/doSearch?Contrib=Menezes%2C+S+M+S

http://informahealthcare.com/action/doSearch?Contrib=Viana%2C+G+S+B

196

Nabati, F., Mojab, F., Habibi-Rezaei, M., Bagherzadeh, K., Amanlou, M., Yousefi, B., 2012.

Large scale screening of commonly used Iranian traditional medicinal plants

against urease activity. DARU Journal of Pharmaceutical Sciences 20, 72.

Naczk, M., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: Occurrence, extraction

and analysis. Journal of Pharmaceutical and Biomedical Analysis 41, 1523-1542.

Nanditha, B., Prabhasankar, P., 2008. Antioxidants in bakery products: a review. Critical

Reviews in Food Science and Nutrition 49, 1–27.

Naveena, B. M., Sen, A. R., Kingsly, R. P., Singh, D. B., Kondaiah, N., 2008a. Antioxidant

activity of pomegranate rind powder extract in cooked chicken

patties. International Journal of Food Science & Technology 43, 1807-1812.

Naveena, B.M., Sen, A.R., Vaithiyanathan, S., Babji, Y., Kondaiah, N., 2008b. Comparative

efficacy of pomegranate juice, pomegranate rind powder extract and BHT as

antioxidants in cooked chicken patties. Meat Science 80, 1304–1308.

Naz, S., Siddiqi, R., Ahmad, S., Rasool, S.A., and Sayeefd, S.A., 2007. Antibacterial activity

directed isolation of compounds from Punica granatum. Journal of Food

Science72, 341–345.

Nazni, P., Pradheepa, S., Hasan, A., 2010. Effects of weaning biscuits on the nutritional profile

and the cognitive development in preschool children. Italian Journal of Pediatrics

36, 1–6.

Negi, P., Jayaprakasha, J., 2003. Antioxidant and antibacterial activities of Punica granatum peel

extracts. Journal of Food Science68, 1473–1477.

Negi, P.S., Jayaprakasha, G.K., Jena, B.S., 2003. Antioxidant and antimutagenic activities of

pomegranate peel extracts. Food Chemistry 80, 393-398.

Nemeth, N., Alexy, T., Furka, A., Baskurt, O. K., Meiselman, H. J., Furka, I., Miko, I., 2009.

Inter-species differences in hematocrit to blood viscosity ratio. Biorheology 46,

155-165.

Neyrinck, A.M., Van Hee, V.F., Bindels, L.B., De Backer, F., Cani, P.D., Delzenne, N.M., 2012.

Polyphenol-rich extract of pomegranate peel alleviates tissue inflammation and

hypercholesterolaemia in high-fat diet-induced obese mice: potential implication

of the gut microbiota. British Journal of Nutrition 7, 1–8.

197

Nonaka, G., Nishioka, I., Nishizawa, M., Yamagishi, T., Kashiwada, Y., Dutschman, G.E.,

Bodner, A.J., Kilkuskie, R.E., Cheng, Y.C., Lee, K.H., 1990. Anti-AIDS agents,

2: Inhibitory effects of tannins on HIV reverse transcriptase and HIV replication

in H9 lymphocyte cells. Journal of Natural Products 53, 587-595.

Official Methods of Analysis of AOAC International, 2000. Official Methods of Analysis.

Official Method 999.11, 17th Ed., AOAC International, Gaithersburg, MD.

Ogunlana, Olujoke, O., 2012. Phytochemical, biological and toxicological studies of the extracts

of young twigs and leaves of gray nicker nut (Caesalpinia bonduc (Linn)) Roxb.

Diss. Department of Biological Sciences, College of Science and Technology,

Covenant University, OTA.

Okonogi, S., Duangrat, C., Anuchpreeda, S., Tachakittirungrod, S., Chowwanapoonpohn, S.,

2007. Comparison of antioxidant capacities and cytotoxicities of certain fruit

peels. Food Chemistry 103, 839-846.

Okuda, T., 1999. Novel aspects of tannins—renewed concepts and structure-activity

relationships. Current Organic Chemistry 3, 609-622.

Okuda, T., 2005. Systematics and health effects of chemically distinct tannins in medicinal

plants. Phytochemistry 66, 2012-2031.

Opara, L. U., Al-Ani, M. R., Al-Shuaibi, Y. S., 2009. Physico-chemical properties, vitamin C

content, and antimicrobial properties of pomegranate fruit (Punica granatum L.).

Food and Bioprocess Technology 2, 315-321.

Orgil, O., Schwartz, E., Baruch, L., Matityahu, I., Mahajna, J., Amir, R., 2014. The antioxidative

and anti-proliferative potential of non-edible organs of the pomegranate fruit and

tree. LWT - Food Science and Technology 58, 571-577.

Ouachrif, A., Khalki, H., Chaib, S., Mountassir, M., Aboufatima, R., Farouk, L., Benharraf, A.,

Chait, A., 2012. Comparative study of the anti-inflammatory and antinociceptive

effects of two varieties of Punicagranatum. Pharmaceutical Biology 50, 429-438.

Ozdemir, H., Soyer, A., Tagi, S., Turan, M., 2014. Effects of antimicrobial and antioxidant

activity of pomegranate peel extract on the quality of beef meatballs. GIDA-J

Food 39, 355-362.

198

Pacheco-Palencia, L.A., Noratto, G., Hingorani, L., Talcott, S.T., Mertens-Talcott, S.U., 2008.

Protective effect of standardized pomegranate (PunicagranatumL.) polyphenolic

extract in ultraviolet irradiated human skin fibroblasts. Journal of Agricultural and

Food Chemistry 56, 8434-8441.

Pai, V., Chanu, T.R., Chakraborty, R., Raju, B., Lobo, R., Ballal, M., 2011. Evaluation of the

antimicrobial activity of Punica granatum peel against the enteric pathogens: An

in vitro study. Asian Journal of Plant Science and Research 1, 57-62.

Panichayupakaranant, P., Itsuriya, A., Sirikatitham, A., 2010a. Preparation method and stability

of ellagic acid-rich pomegranate fruit peel extract. Pharmaceutical Biology 48,

201-205.

Panichayupakaranant, P., Tewtrakul, S., Yuenyongsawad, S., 2010b. Antibacterial,

antiinflammatory and antiallergic activities of standardized pomegranate rind

extract. Food Chemistry 123, 400-403.

Patel, C., Dadhaniya, P., Hingorani, L., Soni, M. G., 2008. Safety assessment of pomegranate

fruit extract: acute and subchronic toxicity studies. Food and Chemical

Toxicology 46, 2728-2735.

Pearson, T.A., Blair, S.N., Daniels, S.R., Eckel, R.H., Fair, J.M., Fortmann, S.P., Franklin, B.A.,

Goldstein, L.B., Greenland, P., Grundy, S.M., Hong, Y., 2002. AHA guidelines

for primary prevention of cardiovascular disease and stroke: 2002 update

consensus panel guide to comprehensive risk reduction for adult patients without

coronary or other atherosclerotic vascular diseases. Circulation 106, 388-891.

Peters Jr, T., 1995. All about albumin: biochemistry, genetics, and medical applications (1st ed).

Academic Press: New York.

Popov-Raljic, J.V., Mastilovic, J.S., Lalicic-Petronijevic, J.G., Kevresan, Z.S., Demin, M.A.,

2013. Sensory and color properties of dietary cookies with different fiber sources

during 180 days of storage. Hemijska Industrija 67, 123-134.

199

Prentice, A., Bates, C.J., 1993. An appraisal of the adequacy of dietary mineral intakes in

developing countries for bone growth and development in children. Nutrition

Research Reviews 6,51–69.

Qu, W., Breksa III, A. P., Pan, Z., Ma, H., Mchugh, T. H., 2012. Storage stability of sterilized

liquid extracts from pomegranate peel. Journal of Food Science 77, C765-C772.

Qu, W., Pan, Z., Ma, H., 2010. Extraction modeling and activities of antioxidants from

pomegranate marc. Journal of Food Engineering 99, 16-23.

Quigley, E.M., 2010. Prebiotics and probiotics; modifying and mining the microbiota.

Pharmacological Research61, 213–218

Ramadan, Afaf-haniem, El-badrawey, S., Abd el-ghany, M., Nagib, R.M., 2010. Utilization of

hydro-alcoholic extracts for peel and rind and juice of pomegranate as natural

antioxidants in cotton seed oil. The 5thArab and 2nd International Annual

Scientific Conference, Faculty of Specific Education Mansoura University - Egypt

April, 8-9, 2009. pp. 2442–2462.

Ramzi, D. O. M., 2016. Effect of different ratios of pomegranate peels on hematological,

biochemical parameters and reproductive hormones of Karadi ram lambs.

International Journal of Veterinary Science 5, 19-23.

Ramzi, D.O.M., 2016. Effect of different ratios of pomegranate peels on hematological,

biochemical parameters and reproductive hormones of karadi ram lambs.

International Journal of Veterinary Science 5, 19-23.

Rathi, P., Rajput, C.S., Singhal, S., 2014. Evaluation of total phenolic contents, antioxidant and

antibacterial capacity of aqueous methanolic extracts obtained from punica

granatum peel. International Journal of Pharmaceutical Sciences Review and

Research 25, 92

Reddy, M., Gupta, S., Jacob, M., Khan, S., Ferreira, D., 2007. Antioxidant, antimalarial and

antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids

from PunicagranatumL. Planta Medica73, 461–467.

Reddy, V., Urooj, A., Kumar, A., 2005. Evaluation of antioxidant activity of some plants extracts

and their application in biscuits. Food Chemistry 90, 317–321.

200

Romier, B., Van De Walle, J., During, A., Larondelle, Y., Schneider, Y.J., 2008. Modulation of

signalling nuclear factor-kappaB activation pathway by polyphenols in human

intestinal Caco-2 cells. The British Journal of Nutrition 100, 542-551.

Rommel, A., Ronald, E., 1993. Ellagic acid content of red raspberry juice as influenced by

cultivar, processing, and environmental factors. Journal of Agricultural and Food

Chemistry4, 1951–1960.

Rosenblat, M., Aviram, M., 2009. Paraoxonases role in the prevention of cardiovascular

diseases. Biofactors 35, 98-104.

Rosenblat, M., Volkova, N., Coleman, R., Aviram, M., 2006. Pomegranate byproduct

administration to apolipoprotein e-deficient mice attenuates atherosclerosis

development as a result of decreased macrophage oxidative stress and reduced

cellular uptake of oxidized low-density lipoprotein. Journal of Agricultural and

Food Chemistry 54, 1928-1935.

Rummun, N., Somanah, J., Ramsaha, S., Bahorun, T., Neergheen-Bhujun, V. S., 2013.

Bioactivity of nonedible parts of Punica granatum L.: a potential source of

functional ingredients. International Journal of Food Science, 2013.

Rupasinghe, H.V., Wang, L., Huber, G.M., Pitts, N.L. 2008. Effect of baking on dietary fiber and

phenolics of muffins incorporated with apple skin powder. Food Chemistry 107,

1217-1224.

Saad, H., Charrier-El Bouhtoury, F., Pizzi, A., Rode, K., Charrier, B., Ayed, N., 2012.

Characterization of pomegranate peels tannin extractives. Industrial Crops and

Products 40, 239 – 246.

Sadeghian, A., Ghorbani, A., Mohamadi-Nejad, A., Rakhshandeh, H., 2011. Antimicrobial

activity of aqueous and methanolic extracts of pomegranate fruit skin. Avicenna

Journal of Phytomedicine 1, 67-73.

Sadeghipour, A., Eidi, M., Ilchizadeh Kavgani, A., Ghahramani, R., Shahabzadeh, S., Anissian,

A., 2014. Lipid lowering effect of Punica granatum L. peel in high lipid diet fed

male rats. Evidence-Based Complementary and Alternative Medicine, 2014.

201

Saeed, M., Nadeem, M., Khan, M.R., Asim, M., Shabbir, A.S., Amir, R.M., 2013. Antimicrobial

activity of Syzygium aromaticum extracts against food spoilage bacteria. African

Journal of Microbiological Research 7, 4848-4856.

Saranraj, P., 2012. Microbial spoilage of bakery products and its control by preservatives. Int. J.

Pharm. Biology Archives3, 38-48.

Sartippour, M.R., Seeram, N.P., Rao, J.Y., Moro, A., Harris, D.M., Henning, S.M., Firouzi, A.,

Rettig, M.B., Aronson, W.J., Pantuck, A.J., Heber, D., 2008. Ellagitannin-rich

pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo.

International Journal of Oncology 32, 475-480.

Sarwar, N., Danesh, J., Eiriksdottir, G., Sigurdsson, G., Wareham, N., Bingham, S., Boekholdt,

S.M., Khaw, K.T., Gudnason, V., 2007. Triglycerides and the risk of coronary

heart disease 10 158 incident cases among 262 525 participants in 29 western

prospective studies. Circulation 115, 450-458.

Sayed-Ahmed, E.F., 2014. Evaluation of pomegranate peel fortified pan bread on body weight

loss. International Journal of Nutrition and Food Sciences 3, 411 – 420.

Scalbert, A., Deprez, S., Mila, I., Albrecht, A.M., Huneau, J.F., Rabot, S., 2000.

Proanthocyanidins and human health: systemic effects and local effects in the

gut. Biofactors 13, 115-120.

Scharer, K., 1977. The effect of chronic underfeeding on organ weights of rats How to interpret

organ weight changes in cases of marked growth retardation in toxicity

tests?. Toxicology 7, 45-56.

Seeram, N. P., Heber, D., 2011. Purification of pomegranate ellagitannins and their uses thereof.

US patents, no. US 7919636 B2.

Seeram, N.P., Adams, L.S., Henning, S.M., Niu, Y., Zhang, Y., Nair, M.G., Heber, D., 2005. In

vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic

acid and a total pomegranate tannin extract are enhanced in combination with

other polyphenols as found in pomegranate juice. The Journal of Nutritional

Biochemistry 16, 360-367.

202

Seeram, N.P., Lee, R., Heber, D., 2004. Bioavailability of ellagic acid in human plasma after

consumption of ellagitannins from pomegranate (Punica granatum L.) juice.

Clinica Chimica Acta; International Journal of Clinical Chemistry 348, 63-68.

Şen, V., Bozkurt, M., Söker, S., Ece, A., Güneş, A., Uluca, Ü., Söker, M., Yel, S., Kaplan, İ.,

2014. The effects of pomegranate and carvacrol on methotrexate-induced bone

marrow toxicity in rats. Clinical & Investigative Medicine 37, 93-101.

Sestili, P., Martinelli, C., Ricci, D., Fraternale, D., Bucchini, A., Giamperi, L., Curcio, R.,

Piccoli, G., Stocchi, V., 2007. Cytoprotective effect of preparations from various

parts of Punica granatum L. fruits in oxidatively injured mammalian cells in

comparison with their antioxidant capacity in cell free systems. Pharmacological

Research : the Official Journal of the Italian Pharmacological Society 56, 18-26.

Shahidi, F., Janitha, P. K., Wanasundara, P. D. (1992). Phenolic antioxidants. Critical Reviews in

Food Science & Nutrition 32, 67-103

Shema-Didi, L., Sela, S., Ore, L., Shapiro, G., Geron, R., Moshe, G., Kristal, B., 2012. One year

of pomegranate juice intake decreases oxidative stress, inflammation, and

incidence of infections in hemodialysis patients: a randomized placebo-controlled

trial. Free Radical Biology and Medicine 53, 297-304.

Sies, H., 1997. Physiological society symposium: impaired endothelial and smooth muscle cell

function in oxidative stress oxidative stress: oxidants and antioxidants.

Experimental Physiology 82, 291-295.

Singh, D.B., Kingsly, A.R.P., Meena, H.R., Kaur, N., 2007.Studies on physico-chemical and

nutritional properties of pomegranate rind powder. Indian Journal of

Horticulture64, 155–158.

Singh, R.P., Murthy, C.K.N., Jayaprakasha, G.K., 2002. Studies on the antioxidant activity of

pomegranate (Punica granatum) peel and seed extracts using in vitro models.

Journal of Agricultural and Food Chemistry, 50, 81-86.

Sloan, F.A., Gelband, H., 2007. Cancer control opportunities in low- and middle-income

countries. Institute of Medicine of the National Academies, National Academies

Press, Washington, DC

203

Sowmya Kote, D., Sunder Kote, D., 2011. Effect of pomegranate juice on dental plaque

microorganisms (streptococci and lactobacilli). Ancient Science of Life, 31, 49-

51

Steel, R.G.D., Torrie, J.H., Dickey, D. 1997. Principals and procedures of statistics (3rd Ed.),

McGraw Hills, New York.

Sumbul, S., Ahmad, M.A., Mohd, A., Mohd A., 2011. Role of phenolic compounds in peptic

ulcer: An overview. Journal of Pharmacy and Bioallied Sciences 3, 361-367.

Summers, C., Rankin, S. M., Condliffe, A. M., Singh, N., Peters, A. M., Chilvers, E. R., 2010.

Neutrophil kinetics in health and disease. Trends in Immunology 31, 318-324.

Sun, B., Ricardo-Da-Silva, J.M., Spranger, I. 1998. Critical factors of vanillin assay for catechins

and proanthocyanidins. Journal of Agricultural and Food Chemistry 46, 4267-

4274.

Sundararajan, A., Ganapathy, R., Huan, L., Dunlap, J.R., Webby, R.J., Kotwal, G.J., Sangster,

M.Y., 2010. Influenza virus variation in susceptibility to inactivation by

pomegranate polyphenols is determined by envelope glycoproteins. Antiviral

Research 88, 1-9.

Taguri, T., Tanaka, T., Kouno, I., 2004. Antimicrobial activity of 10 different plant polyphenols

against bacteria causing food borne disease. Biological and Pharmaceutical

Bulletin27, 1965–1969.

Tasaki, M., Umemura, T., Maeda, M., Ishii, Y., Okamura, T., Inoue, T., Kuroiwa, Y., Hirose,

M., Nishikawa, A., 2008. Safety assessment of ellagic acid, a food additive, in a

subchronic toxicity study using F344 rats. Food and Chemical Toxicology 46,

1119-1124.

Tayel, A. A., El‐Tras, W. F., 2010. Anticandidal activity of pomegranate peel extract aerosol as

an applicable sanitizing method. Mycoses, 53, 117-122.

Techathuvanan, C., Reyes, F., David, J. R., Davidson, P. M., 2014. Efficacy of commercial

natural antimicrobials alone and in combinations against pathogenic and spoilage

microorganisms. Journal of Food Protection 77, 269-275.

204

Tezcan, F., Gultekin-Ozguven, M., Diken, T., Ozcelik, B., Erim, F.B., 2009. Antioxidant activity

and total phenolic, organic acid, and sugar content in commercial pomegranate

juices. Food Chemistry 115, 873–877.

Torronen, R., 2009. Sources and health effects of dietary ellagitannins.Chemistry and Biology of

Ellagitannins, An Underestimated Class of Bioactive Plant Polyphenols, 298-315.

Touchburn, S., Simon, J., Leclercq, B., 1981. Evidence of a glucose-insulin imbalance and effect

of dietary protein and energy level in chickens selected for high abdominal fat

content. The Journal of Nutrition111, 325-335.

Tugcu, V., Kemahli, E., Ozbek, E., Arinci, Y. V., Uhri, M., Erturkuner, P., Metin, G., Seckin, I.,

Karaca, C., Ipekoglu, N., Altug, T., 2008. Protective effect of a potent

antioxidant, pomegranate juice, in the kidney of rats with nephrolithiasis induced

by ethylene glycol. Journal of Endourology 22, 2723-2732.

Turksoy, S., Ozkaya, B., 2011. Pumpkin and carrot pomace powders as a source of dietary fiber

and their effects on the mixing properties of wheat flour dough and cookie

quality. Food Science and Technology Research17, 545–553.

Tyagi, V.K., Vasishtha, A.K., 1996. Changes in characteristics and composition of oils during

deep-fat frying. Journal of American Oil Chemists Society 73, 499 – 506.

Ullah, N., Ali, J., Khan, F. A., Khurram, M., Hussain, A., Rahman, I. U., 2012. Proximate

composition, minerals content, antibacterial and antifungal activity evaluation of

pomegranate (Punica granatum L.) peels powder. Middle-East Journal of

Scientific Research 11, 396-401.

Uysal, H., Bilgicili, N., Elgun, A., Ibanoglu, S., Herken, E.N., Demir, M.K., 2007. Effect of

dietary fiber and xylanase enzyme addition on the selected properties of wire cut

cookies. Journal of Food Engineering 78, 1074 – 1078.

Vaher, M., Matso, K., Levandi, T., Helmja, K., Kaljurand, M., 2010. Phenolic compounds and

the antioxidant activity of the bran, flour and whole grain of different wheat

varieties. Procedia Chemistry 2, 76 – 82.

Van Acker, S. A., Tromp, M. N., Griffioen, D. H., Van Bennekom, W. P., Van Der Vijgh, W. J.,

Bast, A. 1996. Structural aspects of antioxidant activity of flavonoids. Free

Radical Biology and Medicine 20, 331-342.

http://link.springer.com/search?facet-author=%22A.+K.+Vasishtha%22

205

Vasconcelos, L.C., Sampaio, M.C., Sampaio, F.C., Higino, J.S., 2003. Use of Punica granatum

as an antifungal agent against candidosis associated with denture stomatitis.

Mycoses 46, 192-196.

Vennat, B., Bos, M., Pourrat, A., Bastide, P., 1994. Procyanidins from tormentil: fractionation

and study of the anti-radical activity towards superoxide anion. Biological and

Pharmaceutical Bulletin 17, 1613-1615.

Ventura, J., Alarcon-Aguilar, F., Roman-Ramos, R., Campos-Sepulveda, E., Reyes-Vega, ML.,

Boone-Villa, V.D., Jasso-Villagomez, E.I., Aguilar, C.N., 2013. Quality and

antioxidant properties of a reduced-sugar pomegranate juice jelly with an aqueous

extract of pomegranate peels. Food Chemistry 136,109–115.

Vidal, A., Fallarero, A., Pena, B.R., Medina, M.E., Gra, B., Rivera, F., Gutierrez, Y., Vuorela,

P.M., 2003. Studies on the toxicity of Punica granatum L. (Punicaceae) whole

fruit extracts. Journal of Ethnopharmacology 89, 295–300.

Viuda-Martos, M., Fernandez-Lopez, J., Perez-Alvarez, J.A., 2010. Pomegranate and its many

functional components as related to human health: A review. Comprehensive

Reviews in Food Science and Food Safety9, 635–647.

Viuda-Martos, M., Ruiz-Navajas Y., Martin-Sanchez A., Sanchez-Zapata E., Fernandez- Lopez

J., Sendra, E., Sayas-Barbera, E., Navarro, C., Perez-Alvarez, J.A., 2012.

Chemical, physico-chemical and functional properties of pomegranate (Punica

granatum L.) bagasses powder co-product. Journal of Food Engineering 110, 220-

224.

Viuda-Martos,M., Perez-Alvarez, J.A., Sendra, E., Fernandez-Lopez, J., 2013. In vitro

antioxidant properties of pomegranate (Punica granatum) peel powder extract

obtained as coproduct in the juice extraction process. Journal of Food Processing

and Preservation. 37, 772-776.

Voravuthikunchai, S.P., Sririrak, T., Limsuwan, S., Supawita, T., Iida, T, Honda, T., 2005.

Inhibitory effects of active compounds from Punica granatum pericarp on

Verocytotoxin by enetrohaemorrhagic Escherichia coli 0157: H7. Journal of

Health Science 51, 590-596.

http://www.sciencedirect.com/science/article/pii/S030881461201151X









http://www.sciencedirect.com/science/journal/03088146/136/1

206

Weaver, C.M., Proulx, W.R., Heaney, R., 1999. Choices for achieving adequate dietary calcium

with a vegetarian diet. American Journal of Clinical Nutrition 70, 543–548.

Wiseman, H., Halliwell, B., 1996. Damage to DNA by reactive oxygen and nitrogen species:

role in inflammatory disease and progression to cancer.Biochemical

Journal 313(Pt 1), 17.

Wong, S. P., Leong, L. P., Koh, J. H. W., 2006. Antioxidant activities of aqueous extracts of

selected plants. Food Chemistry 99, 775-783.

World Food Program (WFP). 2013. Special nutritional products. Available at:

http://www.wfp.org/nutrition/special-nutritional-products. Accessed on 29 June

2013.

Yoshida, T., Hatano, T., Ito, H., 2000. Chemistry and function of vegetable polyphenols with

high molecular weights. Biofactors 13, 121-125.

Yoshida, T., Hatano, T., Ito, H., Okuda, T., 2009. Structural diversity and antimicrobial activities

of ellagitannins. Chemistry and biology of ellagitannins. An underestimated class

of bioactive plant polyphenols. World Scientific, Singapore, 55-93.

Yoshimura, M., Watanabe, Y., Kasai, K., Yamakoshi, J., Koga, T., 2005. Inhibitory effect of an

ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-

induced pigmentation. Bioscience, Biotechnology, and Biochemistry, 69, 2368-

2373.

Youssef, H..M.., Mousa, R.M.A., 2012. Nutritional assessment of wheat biscuits and fortified

wheat biscuits with citrus peel powder. Food and Public Health 2,55–60.

Zahin, M., Aqil, F., Ahmad, I., 2010. Broad spectrum antimutagenic activity of antioxidant

active fraction of punica granatum L. peel extracts. Mutation Research 703, 99-

107

Zaias, J., Mineau, M., Cray, C., Yoon, D., Altman, N. H., 2009. Reference values for serum

proteins of common laboratory rodent strains. Journal of the American

Association for Laboratory Animal Science 48, 387-390.

Zam, W., Bashour, G., Abdelwahed, W., Khayata, W., 2012. Separation and purification of

proanthocyanidins extracted from pomegranate’s peels (Punica granatum).

http://www.wfp.org/nutrition/special-nutritional-products

207

International Journal of Pharmaceutical Sciences and Nanotechnology3, 1808-

1813.

Zhao, X., Yuan, Z., Fang, Y., Yin, Y., Feng, L.2013. Characterization and evaluation of major

anthocyanins in pomegranate (Punica granatum L.) peel of different cultivars and

their developmental phases. European Food Research &. Technology 236, 109-

117.

i

Appendix- I

Estimated daily consumption of PoPx

Animals avg. wt. during the study: 201

Daily average diet consumption: 20g

20g Diet carries extracts amount:

0.25% = 50mg

0.50% = 100mg

1.0% = 200mg

On an average daily PoPx consumption per kg B.W

0.25% = 248.8mg/kg b.w./day

0.50% = 497.5 mg/kg b.w./day

1.0% = 995.0 mg/kg b.w./day

ii

Appendix- II

SENSORY EVALUATION PROFORMA

(Score Sheet)

Project Title: Pomegranate peel base novel food product

Product (s): Pomegranate peel, peal meal and peel extracts supplemented cookies

Name of the judge: ___________________________________

Date: ___________________________________

Treatment Color Color Crispiness Texture Overall

acceptability

Signature: ______________

Instructions for the Judges:

Please examine the sample for the stated quality aspects using following scale:-

1 Dislike extremely

2 Dislike very much

3 Dislike moderately

4 Dislike slightly

5 Neither like nor dislike

6 Like slightly

7 Like moderately

8 Like very much

9 Like extremely

iii

Appendix- III

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v

vi

vii

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