Physico-Chemical Characterization and Biological Activity of Daucus Carota Cultivars Indigenous to Pakistan

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PHYSICO-CHEMICAL CHARACTERIZATION AND BIOLOGICAL ACTIVITY OF DAUCUS CAROTA CULTIVARS INDIGENOUS TO PAKISTAN

Nadeem Abbas Faisal 2011-GCUF-05654

Thesis submitted in partial fulfillment of the Requirements for the degree of

DOCTORATE OF PHILOSPHY IN APPLIED CHEMISTRY

DEPARTMENT OF APPLIED CHEMISTRY GOVERNMENT COLLEGE UNIVERSITY FAISALABAD

DEDICATION

This thesis is dedicated to:

The sake of Allah, my Creator and my Master

My great teacher and messenger, Muhammed (PBUH), who taught us the purpose of life.

My academic teachers, they have made a positive difference in my life

My great parents, who never stop giving of themselves in countless ways.

My dearest wife, who leads me through the valley of darkness with light of hope and support.

My beloved kids, Usman and Hasnat, whom I can't force myself to stop loving.

DECLARATION

The work reported in this thesis was carried out by me under the supervision of Dr. Shahzad Ali Shahid Chatha, Associate Professor, Department of Chemistry, Government College University Faisalabad, Pakistan. I hereby declare that the title of review “Physico-Chemical Characterization and Biological Activity Of Daucus Carota Cultivars Indigenous To Pakistan” and the contents of the this thesis are the product of my own research and no part has been copied from any published source (except the references). I further declare that this work has not been submitted for award of any other degree/diploma. The University may take action if the information provide is found inaccurate at any stage.

______Nadeem Abbas Faisal 2011-GCUF-05654

CONTENTS

List of Tables vi List of Figures viii Acknowledgments ix Chapter 1 INTRODUCTION 1 Chapter 2 REVIEW OF LITERATURE 6 2.1 Traditional uses 7 2.2 Phytochemistry 8 2.3 Carotenoids 11 2.4 Carbohydrates /Sugars 13 2.5 Phenolic acids 18 2.6 Volatiles 25 2.7 Nutritional aspects and potential as functional foods 27 2.8 Pharmacological aspects/health benefits 29 2.8.1 Antioxidant potential 30 2.8.2 Anti-Inflammatory potential 33 2.8.3 Anti-cancer potential 34 2.8.4 Protective potential for cardiovascular diseases 36 2.8.5 Hepatoprotective, wound healing, anti-bacterial and 37 antiviral activities Chapter 3 MATERIALS AND METHODS 40 3.1 Materials 40 3.1.1 Chemicals and reagents 40 3.1.2 Standard compounds 40 3.1.3 Instruments 41 3.1.4 Collection of samples 42 3.1.5 Preparation of the plant material for chemical analysis 42 3.3 Experimental protocol 42 3.3.1 Proximate analysis of carrot cultivars 42 3.3.2 Estimation of physico-chemical properties of carrot seed oil 48 3.3.3 Estimation of physico-chemical properties of D. carota juice 51

3.4 Chromatographic analysis 52 3.4.1Phenolic acid profile by HPLC 52 3.4.1.1 Sample preparation for the determination of 52 phenolic compounds by HPLC 3.4.1.2 Exploration of phenolic compounds by HPLC 53 3.4.2 Saccharides (sugars) profile by HPLC 53 3.5 Spectrophotometric analysis 55 3.5.1 Sample preparation for β carotenes analysis 55 3.5.2 Determination of β carotenes by spectrophotometer 56 3.5.3 Determination of total phenolic (TP) 56 3.5.4 Determination of total ascorbic acid (TAA) 56 3.5.5 Biological activities 57 3.6 A study for the effectiveness of carrot juice on blood parameters 59 3.6.1 Extraction of D. carota juice 59 3.6.2 Subjects 60 3.6.3 Experimental groups 60 3.6.4 Collection of blood samples 60 3.6.5 Blood analysis 60 3.6.6 Total antioxidant status 61 3.6.7 Malondialdehyde status 61 3.7 Statistical analysis 61 Chapter 4 RESULTS AND DISCUSSION 62 4.1 Proximate composition 62 4.1.1 Proximate composition of seeds 62 4.1.2 Proximate composition of carrot roots 64 4.1.3 Mineral profiles of selected cultivars of carrot 67 4.1.4 Proximate composition of top whole of D. carota cultivars 69 4.1.5 Physcico-chemical properties of carrot seed oil 73 4.2 Quantification of individual compounds 78 4.2.1 Quantification of individual sugar compounds 78 4.2.2 Quantification of individual phenolic compounds in 99

different cultivars of D. carota roots 4.2.3 Quantification of β-carotene 100 4.3 Antioxidant activities 113 4.3.1 Antioxidant activities of D. carota roots 113 4.3.2 Antioxidant activities of dehydrated D. carota top whole 116 4.3.3 Antioxidant activities of seeds extract D. carota 122 4.4 Physical analysis of carrot juice 127 4.5 Effect of carrot juice on human blood 130 Chapter 5 SUMMARY 138 Conclusion 141 References 143

LIST OF TABLE

Serial Title Page No. No.

2.1 Traditional uses of carrot 9 2.2 Carotenoids content in carrots of different color. 15 2.3 Comprehensive list of the sugars present in carrots 19 2.4 Comprehensive list of the phenolic acids isolated from Daucus carota 22 2.5 Proximate composition of carrots 31 2.6 Mineral composition of carrots 31 2.7 Vitamin profile of carrots 32 3.1 Description of instruments used throughout the research work 41 3.2 Description of the carrot roots and top whole employed in the current study 43 4.1 Proximate composition of Daucus carota cultivars seeds 66 4.2 Proximate composition Daucus carota cultivars roots 70 4.3 Mineral composition of D. carota roots 71 4.4 Proximate composition of Daucus carota cultivars top whole 76 4.5 Physico-chemical properties of Daucus carota cultivars seed oil 77 4.6 Linear regression parameters obtained from the sugar standards calibration 80 curves. 4.7 Validation parameters for the liquid chromatography method 81

4.8 Sugar recovery rates (standards) added to the D. carota cultivars samples. 82 4.9 Concentration (means ± standard deviation) of sugars in different cultivars of 84 D. carota 4.10 Phenolic acids profile in different cultivars of D. carota roots 101 4.11 β-carotene yield extracted from D. carota cultivars at different temperatures 112 4.12 Total ascorbic acid and total phenolics of selected cultivars of D. carota roots 117 4.13 Antioxidant activities of selected cultivars of D. carota roots 118 4.14 Antioxidant activities of selected cultivars of D. carota top whole 125 4.15 Antioxidant activities of seed extracts from different D. carota cultivars 126 4.16 Physical properties o of D. carota juice collected from selected cultivars of D. 129 carota roots.

Page Serial Titles No. No.

4.17 Physical parameters, blood chemistry, lipid profile, and antioxidant status 134 of volunteers of treatment group No 1 (20 to 30 years) 4.18 Physical parameters, blood chemistry, lipid profile, and antioxidant status 135 of volunteers of treatment group No 2 (31 to 40 years) 4.19 Physical parameters, blood chemistry, lipid profile, and antioxidant status 136 of volunteers of treatment group No 2 (31 to 40 years) 4.20 Physical parameters, blood chemistry, lipid profile, and antioxidant status 137 of volunteers of treatment group No 4 (51 to 60 years)

LIST OF FIGURES

Serial Title Page No. No. 2.1 Structures of some common isomers of carotenoids 14 2.2 The major phenolic compound present in carrots 24 2.3 Structural formulas of terpinolene, falcarinol, falcarindiol, falcarindio 3- 28 acetate 2.4 Photographs of rats showing different stages of wound healing 38 4.1 Chromatogram of sugars profile for mixture of sugars standards 86 4.2 Typical chromatograms of sugar profiles for DCP cultivar 87 4.3 Typical chromatograms of sugar profiles for DC90 cultivar 88 4.4 Typical chromatograms of sugar profiles for DC3 cultivar 89 4.5 Typical chromatograms of sugar profiles for DCW cultivar 90 4.6 Typical chromatograms of sugar profiles for DCY cultivar 91 4.7 Typical chromatograms of sugar profiles for T29 cultivar 92 4.8 Typical chromatograms of sugar profiles for DCW cultivar 93 4.9 Calibration curve for individual maltodextrin at standard working conditions 94 4.10 Calibration curve for individual maltotriose at standard working conditions 95 4.11 Calibration curve for individual maltose at standard working conditions 96 4.12 Calibration curve for individual glucose at standard working conditions 97 4.13 Calibration curve for individual fructose at standard working conditions 98 4.14 HPLC chromatograph mixture of standard phenolic compounds 103 4.15 HPLC chromatograph of phenolic compounds in T29 cultivar 104 4.16 HPLC chromatograph of phenolic compounds in DCY cultivar 105 4.17 HPLC chromatograph of phenolic compounds in DCR cultivar. 106 4.18 HPLC chromatograph of phenolic compounds in DC90 cultivar. 107 4.19 HPLC chromatograph of phenolic compounds in DCP cultivar. 108 4.20 HPLC chromatograph of phenolic compounds in DCW cultivar 109 4.21 HPLC chromatograph of phenolic compounds in DC3 cultivar 110 4.22 Relationship between total antioxidant activity and total phenolic contents in 119 D. carota 4.23 Relationship between DPPH scavenging activity with total phenolic contents 120 in D. carota

ACKNOWLEDGEMENTS

I bow my head before Almighty Allah, The omnipotent, The omnipresent, The merciful, The most gracious, The compassionate, The beneficent, who is the entire and only source of every knowledge and wisdom endowed to mankind and who blessed me with the ability to do this work. It is the blessing of Almighty Allah and His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal. . I would like to take this opportunity to convey my cordial gratitude and appreciation to my worthy, reverently and zealot supervisor Dr. Shahzad Ali Shahid Chatha, Associate Professor, Department of Chemistry, Government College University, Faisalabad, Pakistan. Without whose constant help, deep interest and vigilant guidance, the completion of this thesis was not possible. I am really indebted to him for his accommodative attitude, thought provoking guidance, immense intellectual input, patience and sympathetic behavior. I would like to pay my deepest gratitude and appreciation to one of the member of my supervisory committee Dr. Abdullah Ijaz Hussain, Associate Professor, Department of Chemistry, Government College University, Faisalabad, Pakistan, for her generous cooperation and kind assistance and providing valuable suggestions during accomplishment of my Ph.D. I am thankful to one of the member of my supervisory committee Dr. Ali Imran, Assistant Professor, Department of Food Science and Technology Government College University, Faisalabad , Pakistan, for her generous cooperation and kind assistance and providing valuable suggestions during accomplishment of my Ph.D. I am extremely grateful to Dr. Mateen Abbas, Assistant Professor, Department of Toxicology, Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan. I am also thankful to Dr. Muhammad Ikram, Botoniest, Vegetable section of Ayub Agricultural Research Institute (AARI) Faisalabad, Pakistan for providing me the samples of available cultivars throughout the research. Special thanks to all of my teachers who taught me during my academic career. I am also highly indebted to my best friends and fellows especially, Hafiz Muhammad Asif, Muhammad Kashif Mahmood. Usman Hanif, Riaz Hussain, Imran Mali, Samra Barkaat, Noureen Arshad and rest of my fellows for their assistance, good company, marvelous behavior and friendly attitude.

Last but not least, I really acknowledge and offer my heartiest gratitude to all members of my family especially, father, Faqeer Muhammad, mother, Rashidian BB, wife, Nabeela Nadeem, younger brother, Naeem Abbas, sisters, Najma, Rakshanda, Farkhanda and my beloved sons, Muhammad Usman Faisal and Muhammad Hasnat Faisal for their huge sacrifice, moral support, cooperation, encouragement, patience, tolerance and prayers for my health and success which enabled me to achieve this excellent goal.

Nadeem Abbas Faisal

ABSTRACT

In view of escalating awareness and demand for natural products, having antioxidants in the form of vegetables to get foods with medicinal properties, the research on natural antioxidants has achieved remarkable momentum. Daucus carota is among the emerging vegetables with high economic value, due to craving taste and health promoting attributes. It is extensively acknowledged that cultivar and genotype can affect the physico-chemical characteristics and biological activities in fruits and vegetables. The present project was designed to evaluate compositional variations in newly developed cultivars of carrot indigenous to Pakistan. Investigated cultivars proved to be good source of protein, carbohydrates, oil, crude fiber and minerals with significant (ρ < 0.05) variation. The investigated carrot cultivars showed considerable amounts of phenolic acids and carotenoids with remarkable total antioxidant activity, DPPH scavenging capacity, super radical scavenging capacity and hydroxyl radical scavenging capacity. The composition of individual phenolics and sugars compounds was determined using high performance chromatography (HPLC) with diode array and refractive index detectors, respectively. Among phenolic compounds, 5-caffeoylquinic acid was the major hydroxylcinnamic acid, ranging from 6.98 to 33.23 mg/100g of total phenolic compounds. The total amount of phenolic compounds in DCP cultivar was 54.62 mg/100g, where as the corresponding values in other cultivars ranged from 12.8 to 20.29 mg/100g. Maltose was the predominant sugar in all the seven cultivars of D. carota (1.886-2.463 g/100g), followed by fructose (1.103-2.09 g/100g), glucose (0.963-1.395g/100g). The concentration of maltodextrin and maltotriose ranged from 0.166-0.583 g/100g and 0.004-0.058g/100g respectively in all cultivars of D. carota. A pilot study was conducted to evaluate the effect of consuming 250mL carrot juice for one month on cardiovascular risk markers, body mass index (BMI), blood pressure, blood chemistry, blood cells, lipid profile and antioxidant status in industrial workers of different age groups. Drinking carrot juice showed positive effects on blood chemistry, decreased systolic blood pressure and increased plasma antioxidant activity. Overall, the present study explored that investigated carrot cultivars indigenous to Pakistan are the potent source of

15 minerals, carotenoids, natural sugars and phenolic acids with significant biological activities. It is concluded that all the genotyipical different cultivars with compositional variation, having functional and nutraceutical attributes may be beneficial for health conscious consumers.

Chapter 1 INTRODUCTION Vegetables play an important-role in human-nutrition, having low fats and carbohydrates, with significant amount of proteins, vitamins and fiber contents.These have definite biochemical configuration and well-ordered atomic prearrangement (Willett, 2011), which proved the fact that vegetables have a good contribution to recommended dietary allowance (Potter & Hotchkiss 2012). It has been recommended by various nutritionists that at least five portions fruits and vegetable should be included in human diet (Willett, 2011; James & Emmanuel, 2011). Functional attributes of vegetables are beneficial for controlling body functions, which have positive effect on trading of vegetables in domestic and export markets (Nestle, 2013). Antiseptic, antibacterial and antifungal properties of vegetables have safeguarding properties against diseases and good for health maintenance. Vegetables owing the valuable food ingredients are needed for their body repairing properties. Alkaline reserve of the human body can be maintained by the routine consumption of vegetables (Underwood, 2012). Metabolic activities and production of vitamins are regulated by minerals (Soetanet al., 2010; Potter & Hotchkiss 2012; Abbasi et al., 2015). 25 out of 92 minerals are present in living organisms (Soetan et al., 2010).Vitamins regulate the absorbance of phosphorous and calcium, basic components of teeth, bones, muscles, blood, hair and nerve cells (Potter & Hotchkiss2012). Nervous, metabolic, blood-clotting and endocrine systems are also controlled by vitamins (Huskissonet al., 2007).

Vegetables have antioxidants properties in nature owing the presence of phenolic compounds, beneficial for health and food preservation (Rubatzky et al., 2012). Dietary phenolic compounds in vegetables have potent antioxidant capacities than that of synthetic vitamins (Cartea, et al., 2010). Flavanols are the 2/3 of the dietary phenolic compounds in plants, having effective physiological attributes (Ky, 2013). Thereis a direct liaison among the consumption of phenolic-compounds and diminution of cardiovascular diseases (Dauchet et al., 2006). Phenolic compounds showed a remarkable range of pharmacological and biological capacities (SithrangaBoopathy & Kathiresan 2011). In general perception plants (vegetables and fruits) based phenolic compounds are considered as non-nutritional but biologically active compounds having therapeutic attributes. Effectiveness of therapeutic

17 properties depends upon the concentration of phenolic acids i.e. higher the concentration of phenolic compounds higher will be the scavenging capacities (Gunn, 2013; Faisal et al., 2016). Photosynthetic activity in plants results in the formation of sugars. About 80 to 90% sugars are present in soluble portion of fruits and vegetables at ripening (Moretti et al, 2010; Famiani et al., 2012). Fructose and glucose are the major-sugars present in fruits and vegetables, xylose, a monosaccharide, also found in traces (Dikeman et al., 2004; Lenucci et al., 2008). Some famous phenolics with recognized health potential are given under as;

HO O O HO OH HO O

(S) HO (S) O (R) (R) O OH O (E) HO (Z) OH OH O OH OH Cis-5-caffeoylquinic acid 3-Caffeoylquinic-acid

HO O HO

(S) (R)(R) HO O OH (E) O HO (E) OH

HO OH Caffeic acid 3-O-Feruloylquinic-acid

OH OH

O OH (E) O O

HO O O HO OH (E) (S) O (R) (R()R) O HO O (E) OH HO OH OH 5-p-coumaroylquinic acid 3, 4-dicaffeoylquinic acid

Carrot (Daucus-carota) is a-vegetable which belongs to the family-Apiaceae, harvested in autumn and winter seasons (Dawid et al., 2015). There are two major classes of carrot, “Eastern carrots” and “Western Carrots”. Eastern carrots were cultivated in the beginning of 10th century in central Asia and Afghanistan. Commonly these have branched roots and yellow or purple in color (Rupp, 2011; Engwall et al., 2014; Ihemeje et al., 2015; Denker, 2015).Western carrots are orange to red in color. These have multiple taproots, without branched roots. Primarily Western carrots were cultivated in Holland in 15-16th century. The orange color is due to the presence of carotenes, other colors also do exist as novel cultivars (Rupp, 2011; Navazio, 2012; Denker, 2015). Domestic carrots were derived from wild carrots, native to Asia and Europe. Most probably in the past carrots were cultivated for its seeds and leaves in Persia. Today, the taproot is the most common eaten part of this vegetable (Lum & LeVayer, 2016). Carrot is being used in puddings, salads and, processed foods like concentrate, juices, canned, candies, pickle and dried powder (Szymula et al., 2012) A steady increase in the consumption of carrot has been observed owing to its categorization as a natural-source of antioxidants with high anti-cancer-activity owing to the occurrence of phenolic/carotenoids compounds (Song et al., 2010). Carrot plant approved to be nourishing feed for livestock. Its roots, leaves and stems are highly palatable to farm animals (Saunders, 2012); but, foliage can accumulate lofty quantity of nitrates, due to which eating of leaves by ruminants should be monitored cautiously (Lardy & Anderson, 2009). Carrot-roots are particularly loved for their high betacarotene content and were traditionally utilized as winter nourish for dairy-livestock to create yellow color in margarine and cream (Ikauniece, 2015). Researcher reports point to incorporate D. carota into the diet of dairy cows boost the quantity of vitaminA and fattyacids in their milk (Nalecz-Tarwackaet al., 2003; Klein, 2010).

Today consumers demand high quality and naturally available food with functional attributes at reasonable price (Grunert, 2005). Increased awareness to fitness along with unavailability of functional foods plus strong consumers demand creates the need of natural, fresh and convenience food in the form of vegetables. Scientists are in continuous exertions to develop novel cultivars of fruits and vegetables to attain desirable characteristics through different breeding methods (Tester & Langridge 2010). There are several techniques by

19 which plant breeding can be accomplished, ranging from selecting vegetable with desirable characteristics to complex molecular level (Poehlman, 2013). Plant breeding is in practice worldwide, from individual formers to professional breeders and research centers (Hoffmann et al., 2007). Botanists believe that food security can be ensured by developing novel cultivars of vegetables, having high yield, can adapt variable environments, resistant to diseases and drastic conditions (Plucknett & Smith 2014). To improve the functional qualities of carrot, research work is in progress in agriculture sector (Rakin et al., 2007). Quality of carrot depends upon the cortex size, larger the size of cortex (smaller xylem) higher will be the carrot quality. Some cultivars have no or smaller xylem due the deep penetration of cortex to the inner core (Rubatzky et al., 1999; Christensen & Brandt 2006). Naturally occurring subspecies of wild carrot is “sativus”. Research work on this species is going on to decrease woody core, enhance sweetness and functional properties, leading to the development of novel cultivars (Ihemeje et al., 2015). Considering the importance of novel cultivars of vegetables, recently, AyubAgriculture ResearchInstitute (AARI), Faisalabad,Pakistan has developed a few novel cultivars of D. carota (sativus) as an exertion to formulate healthful constituentshaving significant amount of bioactive and functional compounds to consumers in the form of vegetable. The physical properties of these escalating cultivars ; DCW (Daucus carota with white core and white cortex), DCY (Daucus carota with white core and deep yellow cortex), DCP (Daucus carota with light purple core and purple-black cortex), T29 (Daucus carota with white /red mix core and red cortex), DCR (Daucus carota with red core and red cortex), DC3 (Daucus carota with white core and orange-red cortex) and DC90 (Daucus carota with red core and deep- red cortex) are established. In the present study, proximate composition, minerals profile, β-carotenes, sugars profile, phenolic-acids and antioxidantactivity of recently produced cultivars of carrot were scrutinized. Furthermore, by taking into consideration the significance effects of D. carota, an activity has also been conducted on blood chemistry, lipid profile, antioxidant status and malondialdehyde status of human beings taken from an industry. Detailed information regarding the proximate composition, minerals profile, β-carotenes, sugars profile, phenolic- profiles and biological activity of these cultivars with preferred genetic-architecture is expected to contribute as a referral matter to grow more carrot cultivars affluent in

20 remarkable functionalattributes in toting up to their extra nutritious characteristics. The generated-data regarding such characteristics will be beneficial not only for breeders but also for common and health conscious consumers.

Aims and objectives

The research work presented in this dissertation was executed with following directions;

1- To identify and quantify the high-value nutrients of selected cultivars of Carrots 2- To extract potent antioxidant components and evaluate their antioxidant activities 3- To characterize individual sugars in selected cultivars of carrots by chromatographic techniques 4- To characterize the phenolic acids, carotenoids and minerals in selected cultivars of carrots using spectroscopic and chromatographic techniques 5- To investigate the effectiveness of drinking D. carota juice on BMI and blood chemistry, antioxidant capacity and malondialdehyde production

Chapter 2 REVIEW OF LITERATURE Carrot (Daucus carota) belongs to Apiaceae family, having aromatic plants with hollow stem. Being the 16thlargest family of floweringplants it consists of more than 3,700 species and 434 genera (Stevens, 2001).The most primitive perceived utilization of carrot roots as a vegetable is reported from the 10th century in what is at present form in Afghanistan (Iorizzo et al., 2013). The English word “carrot” was derived from French word “carotte” in 1530 (Hirooka et al., 2014). The Late Latin used the word “carota” derived from Greek word “καρωτόν karōton”. European used the word “Ker”, which means “horn” due to its shape and “mork”, which mean edible root. Generally the word “carrot” which mean “root” is used in most of the languages (Engwall et al., 2014). From the Middle East carrots progressively expand north to Europe and climbed east to China and Japan, most likely by means of the Silk Road (Warren, 2015). From the most prehistoric references, in the Middle East, till the sixteenth century, wild carrots were less refined than current cultivars, having either purple or yellow shades (Brücher, 2012). Orange carrots initially showed genetic variations in sixteenth century. These refined cultivars rapidly spread throughout the world during 20thcentury in developing areas (Rubatzky et al., 1999; Simon, 2000; Navazio, 2012). Carrots do not provide a significant amount of energy as diet but do provide valuable nutrients such as vitamins and various phytochemicals (Hashimoto & Nagayama, 2004). D. carota is a very affluent resource of vitaminA as it contains high content of β- carotenes, precursor to vitamin A. It also supplies good daily value of vitamin K. It is a rich source of potassium as mineral. The major phytochemicals found in carrots include carotednoids, phenolics, anthocyanins, polyacetylenes and some other flavoring compounds (Alasalvar et al., 2001; Faisal et al., 2016). The highest antioxidant capacity is possessed by purple carrots which may be due to anthocyanin content. The main antioxidant found in all carrots is chlorogenic acid (Faisal et al., 2016). Besides this carrots show protective potential against many diseases and possess anti-inflammatory, anti-cancer, protective potential for cardiovascular diseases, haepato-protective, wound healing, anti-bacterial and anti-viral potential (Aberoumand, 2011). WorldHealthOrganization (WHO) reported that about 1.0% disability adjusted life years (a gauge of the prospective life lost due to premature death and the years of creative life lost due to disability) and low consumption of fruit and vegetables

22 result in 2.8% of deaths worldwide. Low consumption of vegetables results in gastrointestinal cancer (14%), ischaemic heart diseases (11%) and 9.0 % of stroke deaths (Sullivan et al., 2000).

2.1 Traditional uses

The data regarding traditional uses of carrot root, seeds and leaves is presented in Table 2.1. Traditionally carrots can be eaten in a variety of ways to get its beneficial attributes. First crop of carrots were cultivated for their seeds and aromatic leaves to get medicated benefits rather than roots (Booth, 1957; Bishayee et al., 1995; Ballmer-Weber et al.,2001;Valero and Salmeron, 2003; Colomboet al.,2011; Lim, 2012). Up to 39.0% of beta-carotene can be released from raw carrots through pulping and cooking in edible oil (Hedren et al., 2002). Alternatively these may be eaten after chopping and boiling, frying or steaming, cooking in stew and soaps. These may be used in the preparation of broths with celery and onion (Nahm Jr, 1982; Axelrod, 1999; McDaniel et al., 2002; Zicker, 2005; Gisslen., 2010). Occasionally green carrots are harvested in immature form and eaten by humans after stir-frying as a leaf vegetable (Brinkman et al., 1942; Gustafsson et al., 1995; Hedren et al., 2002; Belie et al., 2002;Kalia, 2005). It is also cited that green carrots contain health hazardous alkaloids (Brown, 2005). Halwa” a carrot dessert” prepared by cooking grated carrots in milk until whole mixture attain semi-solid form, after that nuts and butter are added (Muhammad Ashraf, 1974; Al‐ Kanhal et al., 1999; Gupta., 2000; Haskell, et al., 2005; Nagla, 2007; Khan, 2014; Raju and Pal, 2104; Sharma et al., 2016). Carrot salads are prepared by cooking grated carrot in mustard seeds and green chillies. Thin strips of carrots can also be added in rice and chutney dishes to make them delicious (Anirudhan & Radhakrishnan, et al., 2008; Chapman., 2009; Gayathri, et al., 2004; Desai et al., 2016). In modern ages baby carrots are available as ready to eat snacks in many supermarkets (Izumi et al., 1996; Bidlack et al., 2011; Hu and Commack, 2011; Gramza-Michałowska, & Człapka-Matyasik, 2011; Dueik et al., 2013; Alam et al., 2013) and are used in the preparation of infant’s food, chips and flakes like potato (Baardseth et al., 1995; Skrede et al., 1997; Sulaeman et al., 2001; Kalia, 2005; Fan et al., 2005; Shyu et al., 2005; Liu-Ping et al., 2005). The sweetness of carrot gives it fruit like properties. Grated carrots are used in the preparation of cakes, puddings and many bakery items to give them functional properties

(Hoch and & Ha, 1986; Kuhn, 1989; Devereux et al., 2003; Trinidad et al., 2003; Gambus et al., 2009; Bidlack et al., 2011; Wan and Hiew, 2016). Carrots can also be used in the preparation of jam with fruits, juices and health drinks, either alone or blended with other fruits or vegetables to enhance deliciousness (Shannon, 1999; Marx et al., 2000; Thürmann et al., 2002; LI et al., 2002; Jan and Masih, 2012; Philip et al., 2012). Carrot curry is the famous dish, available thorough out the world which is prepared by blending curry leaves with caroot to enhance the nutritional properties of both vegetables (Khokhar , 1995; Bhavani, 1996; Kotareddy and Devi1997; Bhavani and Kamini, 1998; Suman et al., 2002). Recently carrot roots are used in a variety of ways in biscuits industries for the proportion of carotenoid and functional biscuits at industrial and domestic scale. The functional properties of biscuits are enhanced by using other ingredients or modern cooking methods. (El-Hag et al., 2001; Suman et al., 2002; Kumari and Grewal, 2007; Gambuś et al., 2009; Mridula, 2011 Gayas et al., 2012; SY, 2014). The antibacterial nature of carrot as a whole proved the fact these can be used for preservation of other food materials like edible oil to stop rancidity (Desobry et al., 1998; Valero et al., 2003; Valero and Frances., 2006; Tongnuanchan and Benjakul,2014). From the data regarding traditional uses of carrot it may be concluded that in the past the layman was also aware of about the medicinal properties of carrot; not only root but also about its seeds and top-whole or leaves. In view of that they used it in the formation of delicious dishes to get its functional attributes. In the modern ages carrots are preserved after drying to get its long-term benefits. Carrot pomace is used in the preparation of biscuits, cakes and other bakery items. Now, with the passage of time, scientists are working to enhance the functional attributes by developing new methods of cooking, frying and mixing with other vegetables. 2.2 Phytochemistry Phytonutrients are secondary metabolites found in plants, having antioxidant potential that have positive impact on human health. The consequence of anti-oxidant constituent in sustaining human healthiness from heart diseases and cancer, intriguing the scientists to design the recipes for food industry to produce functional foods (Velioglu et al., 1998; Robards et al., 1999; Kähkönen et al., 1999).

Table 2.1 Traditional uses of carrot

Part of the plant Traditional use Reference

Leaves, seed and Medicinal use Booth, 1957; Bishayee et al., 1995; Ballmer-Weber et al.,2001;Valero and root Salmeron, 2003; Colomboet al.,2011; Lim, 2012 Leaves Preservation of food Almeida et al., 2009

Root Food Singh et al, 2001; Baardseth et al., 1995; Zareifard et al., 2003; Lepedus et al., 2004; Root Cooking and pulping Brinkman et al., 1942; Gustafsson et al., 1995; Hedren et al., 2002; Belie et al.,

25 2002

Root Pet foods , broths Nahm Jr, 1982; Axelrod, 1999; McDaniel et al., 2002; Zicker, 2005; Gisslen., 2010

Root Salads Priepke et al., 1976; Gupta., 2000; Kalia., 2005; Nipa et al., 2011; Sant'Ana et al.,2012;

Root Added to spicy rice and Muhammad Ashraf, 1974; Al‐ Kanhal et al., 1999; Gupta., 2000; Haskell, et al., Gajar Ka Halwa 2005; Nagla, 2007; Khan, 2014; Raju and Pal, 2104; Sharma et al., 2016

Continue Table 2.1

Root Blended with tamarind to Anirudhan & Radhakrishnan, et al., 2008; Chapman., 2009; Gayathri, et al., 2004; make chutney Desai et al., 2016.

Baby roots Snack food Izumi et al., 1996; Bidlack et al., 2011; Hu and Commack, 2011; Gramza- Michałowska, & Człapka-Matyasik, 2011; Dueik et al., 2013; Alam et al., 2013

Root Chips and flakes Baardseth et al., 1995; Skrede et al., 1997; Sulaeman et al., 2001; Kalia, 2005; Fan et al., 2005; Shyu et al., 2005; Liu-Ping et al., 2005 Root Health drink Shannon, 1999; Marx et al., 2000; Thürmann et al., 2002; LI et al., 2002; Jan and Masih, 2012; Philip et al., 2012

Root Biscuits El-Hag et al., 2001; Suman et al., 2002; Kumari and Grewal, 2007; Gambuś et al., 2009; Mridula, 2011 Gayas et al., 2012; SY, 2014

Root Carrot curry Khokhar , 1995; Bhavani, 1996; Kotareddy and Devi1997; Bhavani and Kamini, 1998; Suman et al., 2002

Root Carrot cake Hoch and & Ha, 1986; Kuhn, 1989; Devereux et al., 2003; Trinidad et al., 2003; Gambus et al., 2009; Wan and Hiew, 2016;

In vitro studies, it is reported that phytonutrients like phenolic acids andcarotenoids compounds might do a considerable role to safeguard biological-systems from negative impact of oxidative-stress (Kalt, 2005). The major phytonutrients found in carrots are carotenoids (Block, 1994) phenolic acids (Babic et al., 1993), polyacetylene compounds (Hansen et al., 2003; Kidmose et al., 2004) and chlorigenic acid (Faisal et al., 2016). High content of β-carotene, ascorbic acid and tocopherol are contained in carrots and it is also known as vitaminized food (Hashimoto & Nagayama, 2004). Carrots are believed to be a functional-food with diverse health-promoting properties because they possess variety of phenolic compounds (Hager & Howard, 2006). 2.3 Carotenoids

Carotenoids is a set of phyto-chemicals that constitute a class,up-toseven hundredorganic- complexes in this natural universe (Britton et al., 2004) and cause pigmentation in many fruits and vegetables. The highest amount of β-carotene is found in carrots than the dark green leafy vegetables (De Pee et al., 1998; Jalal et al., 1998). β-carotene usually exists as crystalline form inside chromoplasts of carrot cells, the proteins and residual membranes are responsible for the formation of such crystals (Ben-Shaul et al., 1968; Hornero-Méndez &Mínguez-Mosquera, 2007). Those which are found in abundance and are often quantified in human-serum include β- cryptoxanthin, lutein,β-carotene, zeaxanthin, and lycopene(Mills et al., 2008). The crystalline-nature of the carotenes in D.carota has a negative effect on its bioavailability (Zhou et al., 1996).The total carotenoids concentration found in the eatable portion of carrot roots varies from 6,000 to 54,800 μg/100 g (Simon & Wolff 1987). Structures of some common isomers of caroteniods are presented in Figure 2.1. D.carota is one of the best sources of betacarotene. The carotenoid concentration of carrots varies from 60--120 mg/100 g, but a few types can have ≥300 mg/100 g (Velišek, 1999). As the orange and yellow fleshed cultivars grow, the concentration of carotene content increased. The cortical region has more carotenes than the core. During past four decades,up to 1000ppm carotenoids have been achieved in carrots through traditional breeding (Dias, 2012a; Dias, 2012b; Simon & Goldman,2007). Carotenoids are the main source of vitamin A in the diet, especially β-carotene (Britton, 1995; Nicolle et al., 2003). Water-soluble red-anthocyanin pigment as well as the water- insoluble red-lycopene pigment, located in some cultivars, does not show pro-vitamin A activity.

Some types of carotenoids are the precursor to retinol compound. Orange red and purple carrots carrots contain α- and β-carotene; lutein prevails in yellow & yellowviolet D.carota; and lycopenecompound preponderates in ruby and the new orange/red-purple carrot. Carotenoids are also responsible for the antioxidant property being scavenger of free radicals (Krinsky, 1989; Palozza & Krinsky, 1992). Commonly, carotenoids in foods are of two types, xanthophylls &carotenes which give prominent red or yellow color and improve the prominence of food. The carotenoid compounds may have acyclic-structure or have a ring consisting of five or six carbons at one or both ends of the molecule (Carle &Schiber, 2001). The importance of carotenoids is not just because of being natural pigments; these are responsible for various biological functions and actions. Carotenoids are found intra-cellularly and they take part in the regulation of gene expression or influence cell functions like platelet activation and inhibition. In human-system, the physiological-actions of α and βcarotenes responsible for the production of vitamin A ranged from 50 to 100% respectively (Panalaks & Murray, 1970; Simpson, 1983) and single molecule of beta-carotene produces 2 molecules of retinol in human- system. The medicinal and biological prospective manifested by the carrots might be accredited to the existence of considerable contents of antioxidant carotenoids predominantly β-carotene. β- carotene compounds behave as a free radical and particularly oxygen scavenger in biological systems, contributes to photoprotection, chemoprevention and improve immune system (Deshpande et al., 1995). Macular portion of human eye have considerable amount of consisted of lutein and thus may play a significant role in healthy eye and safety from age-linked macular-deterioration (Tanumihardjo and Yang, 2005). Lutein is a prominent tint in yellowcarrots ((Grassmann et al., 2007) and its concentration may range from 1.0 to 5.0ppm, but they have very low deliberation of carotenes (2.0-4.0ppm) in contrast to orange-carrots ranged from 95-311ppm (Arscott & Tanumihardjo, 2010). Lutein decreases the hazard of light induced oxidative damage which may results in molecular degradation (Kijlstra et al., 2012). Primarily lycopene carotenoid is responsible for the red color of the carrots. High lycopene content causes the carrots to appear red (Dias, 2012a). Meanwhile anthocyanin-rich carrots are purple (Sun et al., 2009). In white cultivars carotenoids are present in traces. Orange carrot has high-amount of α and βcarotenes and thus proving the fact that it is a rich source of pro-vitamin A (Dias, 2012a; Dias, 2012b). Although lycopene is hydrocarbon even then it does

not show pro-vitamin-A activity and is the more effectual anti-oxidant invitro of all the carotene compounds found in considerable quantity in human beings (Di Mascio et al., 1989) and proposed to be involved in defending process from certain types of cancers (Giovannucci, 2002). Lycopene contents in red colored carrots vary from 50-100ppm (Table 2.2). Table 2.2 revealed that carotenoids composition and concentration reported in different studies taking carrots of different colors. The carrots root from different verities and color showed that carotenoids composition and concentration varied from cultivar to cultivar. Over all beta-carotene concentration is on higher side in all the cultivars except white colored carrots on the other hands alpha-carotene is at the second numeral of carotenes present among the carrot cultivars. It also showed that composition depends upon the environmental conditions, soil chemical composition and fertility of soil. In view of data and discussion related to carotenoids, it is concluded that Beta-carotene, lutein, lycopene are the most common types of carotenoids found in all types of colored carrots. It is also noted that carrots are the rich source of dietary carotenoids which provide health benefits, lowering inflammation, healthy growth, body development, boosting immunity, protecting skin damage reduce the risk of eye problems and many types of cancers. There are two ways through which carotenoids act as powerful antioxidant including inhibit the fats- oxidation and deactivate the singlet oxygen. The presence of carotenoids in carrot proved the fact that carrots have potent antioxidant attributes in nature and have the functional food properties.

2.4 Carbohydrates /Sugars Carbohydrates are the important part of diet of common people all around the world (Willcox et al., 2009). Mostly people think, sugars as human enemy due to unhealthy effects. Their deliberation about sugar is unnecessary as extensive citations are available on the importance of sugars (Bolton-Smith, 1996). Sugars play important role in the development of caries (Fontana & Zero, 2006). Sugars are the important energy source, from food they absorbed in the small intestine to blood stream and travel to cell, hence provide energy for cellular functions (Mahan & Raymond, 2016). Extracted sugars are used in the preparation of foods for improving taste and texture (Drewnowski, 1997). Table sugar is also used in the preparation of oral rehydration solution (ORS), for the prevention diarrhea and vomiting in developing countries (Farthing et al., 2013).

Figure 2.1 Structures of some common isomers of carotenoids

Table 2.2 Content of arotenoids in D. carota (carrot) of dissimilar colour. Carrot ppmTotal Reference Colour α-carotene. β-carotene. Lutein. Lycopene. Orange 22 128 2.6 nr Surles et al., 2004 27 69 0.4 0.6 Sun et al., 2009 40 69 nr nr Alasalvar et al., 2001 10-22 18-38 nr nr Rodriguez-Amaya et al., 2008 47.1 47.5 nr nr EL-Qudah et al., 2009 nr nr nr nr Simon et al., 1989 13-31 32-66 0.6-1.8 nr Nicoli et al., 2004 57-70 45-52 4-5 nr Grassmann et al., 2007 Dark Orange 31 185 4.4 17 Surles et al., 2004 45 113 0.7 0.9 Sun et al., 2009 nr nr nr nr Nicoli et al., 2004 75.8 172 0.7 Nr Alasalvar et al., 2001 nr nr nr nr EL-Qudah et al., 2009 96-192 215-311 1.0 Nr Simon et al., 1989 nr nr nr nr Rodriguez-Amaya et al., 2008 nr nr nr nr Grassmann et al., 2007 Yellow 0.5 1.8 5.1 Not detected Surles et al., 2004 0.2 3.6 2.4 0.04 Sun et al., 2009 nr nr nr nr Alasalvar et al., 2001 Nr 3.3 1.4-2.3 Nr Nicoli et al., 2004 nr nr nr nr Simon et al., 1989 nr nr nr nr EL-Qudah et al., 2009 nr nr 5.0-10 nr Grassmann et al., 2007

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Table 2.2 Red nr nr nr nr Rodriguez-Amaya et al., 2008 1.1 3.4 3.2 61 Surles et al., 2004 0.2 22 0.2 50 Sun et al., 2009 nr 35-40 nr 85-100 Grassmann et al., 2007 Purple

Orange 4 123 11 nr Surles et al., 2004 10 28 1.1 0.2 Sun et al., 2009 nr nr nr nr Rodriguez-Amaya et al., 2008 87 161 nr nr Alasalvar et al., 2001 nr nr nr nr EL-Qudah et al., 2009 nr nr nr nr Alasalvar et al., 2001 62-100 65 8-10 nr Grassmann et al., 2007 Simon et al., 1989 White nr 0.06 0.09 nr Surles et al., 2004 0.05 0.34 1.7 0.04 Sun et al., 2009 nr nr nr nr EL-Qudah et al., 2009 nr nr nr nr Alasalvar et al., 2001 nr nr nr nr Rodriguez-Amaya et al., 2008 nr nr nr nr Simon et al., 1989 nr nr nr nr Grassmann et al., 2007

Fortification of foods is also governed by high concentration of sugars, playing role to fight against nutrient deficiency (Roopchand et al., 2012). Among sugars, glucose is most important, which stores in the liver and muscles as glycogen (Tirone, T. A., & Brunicardi, 2001). Eating vegetable, fruit, dairy products and whole gains provide carbohydrates for glycogen production in liver and muscles in the form of glucose. Among vegetables carrot is the potent source of sugars including glucose (Lee et al., 1998). Sugars content is a remarkable parameter for the determination carrot quality which give sweet taste (Simon et al. 1980). Some of the cited studies did not in agreement about the presence of fructose contents in carrot roots. Sugars are concentrated in the carrots' core; generally those with larger diameters will have a larger core and therefore be sweeter (Vervoort et al., 2012). The sugars-content in D. carota cited by many scientists have been accessible in Table 2.3. As it is identified, glucose and sucrose are the main sugars in carrot and widespread study has been available on their contents in D.carota of many varieties ( Svanberg et al., 1997;Alasalvar et al., 2001) and proposed to many types of dispensation and storage-space surroundings (Nyman et al., 2005; Machewad et al., 2003; Svanberg et al., 1997). Soria et al. (2008) described that the average full content of minor carbohydrates was 17mg/g in raw carrots and 23 mg/g in dehydrated carrots. Mannitol was simply situated in four out of eight carrot samples, with contents considerably lesser than 16 mg/g reported by Souci et al. (2000). Myo-inositol levels (2–10 mg/g) were in the equal range as those described by Clements and Darnell (1980) in raw, frozen and canned carrots (0.1–0.5 mg/g). TerhiSuojala, (2000) studied that sugar contents during harvesting periods were dependent on growing site. The sugar symphony pursued the common patterns detailed in previous works (Goris 1969, Phan & Hsu 1973, Ricardo & Sovia 1974, Nilsson 1987; Suojala, 2000), with an broaden of sucrose amount and sucrose/hexose ratio. During harvesting periods the concentration of fructose and glucose turn out to be lower (Gibon et al., 2004). Carrots store two main types of sugars namely glucose and fructose in the central core. After consumption the carbohydrates from carrot in the small intestine transported to liver for the production of glycogen. Although every part of body requires sugars for energy, nerves, brain and blood cells entirely depend upon glucose to function. In view of above discussion, it may be concluded that carbohydrates are the chief constituent of carrots after

water, which makes carrot attractive and sweet. The sugars concretions in carrots are also sufficient for human body requirement. Therefore it is recommended that carrot consumption is necessary, as it is a natural source of energy having fewer side effects. 2.5 Phenolic acids

Phenolic compounds having single aromatic ring are known as phenolic acids, naturally found in carrots and many other fruits and vegetables. The phenolic acid contents in different varieties of carrot by different scientists are presented in Table 2.4. Chlorogenic acids are the major phenolic compounds present in carrots which are derivative of hydroxyl cinnamicacid created by the esterification of cinnamicacids, like p-coumaric acids, caffeic and ferulic, with (-)quinic acid. The organolepticproperties of fresh and processed carrots are because of these compounds (Rubatzky et al., 1999). Alasalvar et al., (2001) reportedeleven types of phenolic-compounds in golden, white, orange, and violet carrots. Chlorogenic (Figure 2.2) contents differ in different varieties of carrots. Violet, , white, orange and golden carrots contain 54.1 mg/100g, 8.5 mg/100g, 4.5mg/100g and 4.4 mg/100g respectively. Chloro- genic-acid characterizes 42.2 to 61.8% of overall phenolic-compounds determined in miscellaneous tissues of D. carota (Zhang & Hamauzee, 2004). The purple D. carota have been recognized as having greatest concentration of all phenolics and their concentrations followed the same sequence as did the chlorogenic acid concentration (Faisal et al., 2016). Carrots possessed 37.60% as bound phenolic-acids, second after potato which has the highest bounded phenolic-acids concentration. Bounded phenolic-acids are present in esters-form and are coupled with cellwall constuents (Kang et al., 2008). Which provide crosslinking aptitude to give structural-constancy in the cellwall- matrix. Bounded phenolic-acids might stay intact in upper gastrointestinaldigestion process and excreted in colon where they can perform positive health outcomes. Chu et al., (2002) reported that potatoes and D. carota might discharge about half of their phenolic-acids in colon, while most of the other vegetables could release only one-fourth.

Table 2.3 Comprehensive list of the sugars present in carrots Sr. # Sugar types Part of plant Concentration Reference 1 Glucose Carrot core Not define Vervoort et al., 2012 2 Sucrose Root 1.96-4.11 g/100 g Alasalvar et al., 2001 3 Fructose Root 0.58-1.47 g/100 g Alasalvar et al., 2001 4 Glucose Root 0.69-1.77 g/100 g Alasalvar et al., 2001 5 Fructose, glucose & sucrose Root Not define Svanberg et al., 1997

6 Minor saccharides Root 17 to 23 mg/g Soria et al., 2008 7 Mannitol Root 16 mg/g Souci et al., 2000 8 Myo-inositol Root 2–10 mg/g Souci et al., 2000 9 Myo-inositol Frozen Root 0.1–0.5 mg/g Clements & Darnell, 1980

10 Total sugar Root 4.5–7.5% Balvoll et al., 1976; Evers, 1989 ; Hogstad et al., 1997

11 Reducing sugars Root 1.67–3.35% Kaur et al. (1976) 12 Non-reducing sugars Root 1.02–1.18% Kaur et al. (1976) 13 Free reducing sugars Root 6–32% Simon and Lindsay 1983 14 Sucrose, glucose, Root Not define Kalra et al., 1987 xylose and fructose 15 Sucrose Root Not define Rosenfeld, 1998;

16 Sucrose Root Not define Balvoll et al, 1976; Evers, 1989; Hogstad et al., 1997

17 Glucose Root 1.19 Sorensen et al., 1997 18 Fructose Root 0.96 Sorensen et al., 1997 19 Sucrose Root 3.09 Sorensen et al., 1997 20 Total sugars Root 5.24 Sorensen et al., 1997 21 Glucose Root 1.35 Klaiber et al., 2005

22 Fructose Root 1.76 Klaiber et al. , 2005

23 Sucrose Root 5.30 Klaiber et al. , 2005 24 Total sugars Root 8.41 Klaiber et al. , 2005 25 Total sugars Root 5.6 Holland et al., 1991

26 Total sugars Root 4.74 USDA, 2016

Sun et al., (2009) examined the relative antioxidant capacity index of different coloured carrots (RACI) by evaluating both hydro-philic and hydro-phobic extracts. They reported that chloro-genic-acid was the main invitro anti-oxidant in diverse colored D.carota and greater contribution was made by the phenolic-acids to the anti-oxidant-activity than carotenoid-compounds. Phenolicsor polyphenols have become significant because of their antioxidant, free radical trapping, and anticancer activities. They have been reported to possess remarkable properties to fight radicals (free), which may be dangerous to body and food (Nagai et al., 2003). Though no known nutritional function is shown by phenolic compounds yet, they may participate an important function in maintaining human fitness due to their antioxidant property (Holiman et al., 1996) .The phenolic acids among tissues were found to be in this order, peel > phloem > xylem. Although, D. carota peel comprises only 11.0% of carrot (fresh weight), so, 54.10% of the total phenolics are present in peel, while 39.5% are present in the phloem tissue and only 6.4% are present in the xylem tissue which has the lowest concentration among all. Anti-oxi-dant and radical--trapping properties in unlike tissues are directly proportional to phenolic content (Zhang & Hamauzee, 2004). The total phenolic contents in D.carota are 26.60±1.70μg/g (Oviasogie et al., 2009) and total phenolics in purple carrot liquid 772±119 mg/L (Karakaya et al., 2001). Compounds produced by plants as outcome of pathogen contagion or acerbic are expersed as phytoalexins since they may be concerned in a protection mechanism (Gonzalez-Lamothe et al., 2009). Phenolic compounds may help plants to fight against bacterial- and fungal-activity. They assemble up in carrots in reaction to ethylene-exposure, injury and cold, (Rubatzky et al., 1999). Total phenolic-acids (Alasalvar et al., 2005), iso-chlorogenic-, chlorogenic-acid (Hager & Howard, 2006), 5-caffeoylquinic acid (trans), p-hydroxy benzoic-acid & its esters (Babic et al., 1993) boost upif storage done in wounded conditions, after shredding processes, or under-revelation to ethylene. The increase in phenolics contents of freshly-cut D. carota,during storeroom is attributed to increase phenylalanine ammonialyase, a wound- induced enzyme (Arscott & Tanumihardjo, 2010). Over 600 compounds constitute of anthocyanins group which are aqua-soluble tinctures and pass on red, violet, and indigo colors to many fruits, flowers, and grains (Neill & Gould, 2003; Gould 2004).

Table 2.4 Comprehensive list of the phenolic acids isolated from Daucus carota Sr. # Phenolic acid type Part of plant Concentration Reference 1 Total phenolics Root (orange) 16.21 ± 0.21 mg/100 g Alasalvar et al., 2001 2 Total phenolics Root (purple) 74.64 ± 3.32 mg/100 g Alasalvar et al., 2001 3 Total phenolics Root (yellow) 7.72 ± 0.22 mg/100 g Alasalvar et al., 2001 4 Total phenolics Root (white) 8.69 ± 0.24 mg/100 g Alasalvar et al., 2001 5 Total phenolics Root 12.9 mg/ 100g Caetano and Leal, 2006

6 chlorogenic acid Root 712 µg/g Przybylska et al., 2007

7 4-hydroxybenzoic acid Root 1241 µg/g Przybylska et al., 2007

8 Total phenolics Root 2972 µg/g Przybylska et al., 2007

9 chlorogenic acid Juice 194 µg/mL Przybylska et al., 2007

10 caffeic acid Juice 235 µg/mL Przybylska et al., 2007

11 phloridzin Juice 54.3 µg/mL Przybylska et al., 2007

12 phloretin Juice 219 µg/mL Przybylska et al., 2007

13 quercetin Juice 17.3 µg/mL Przybylska et al., 2007

14 Total phenolics Juice 648 µg/mL Przybylska et al., 2007

15 Free-phenolics Root 76.0% Kang et al., 2008 16 Bound phenolics Root 37.6% Kang et al., 2008 17 Total phenolics Root 114-306 mg catechin/Kg Koca Bozalan and Karadeniz, 2011

18 Total phenolics Root ~800.0 (GAE)/100 g Augspole et al., 2012 19 Total phenolics Root (white) 18.0 mg/100 g Leja et al., 2013 20 Total phenolics Root (yellow) 21.5 mg/100 g Leja et al., 2013 21 Total phenolics Root (orange) 29.3 mg/100 g Leja et al., 2013 22 Total phenolics Root (red) 31.0 mg/100 g Leja et al., 2013 23 Total phenolics Root (purple) 245.7 mg/100 g Leja et al., 2013 24 Total phenolics Root (Orange) 9.4 mg GAE/100 g Algarra et al., 2014 25 Total phenolics Black Root (Antonina, 2010) 187.8 mg GAE/100 g Algarra et al., 2014 26 Total phenolics Black Root (Purple Haze, 2011) 492.0 mg GAE/100 g Algarra et al., 2014 27 Total phenolics Root 3.3-19.9 mg/ g Lutz et al., 2015 28 Chlorogenic acid Petioles 6.53-8.49 mg/ g Cwalina-Ambroziak et al., 2014 29 Total phenolics Root (T29) 19.71±0.08 mg/100g Faisal et al., 2016 30 Total phenolics Root (DCR) 17.07±0.10 mg/100g Faisal et al., 2016 31 Total phenolics Root (DC3) 20.29±0.08 mg/100g Faisal et al., 2016 32 Total phenolics Root (DC90) 18.72±0.09 mg/100g Faisal et al., 2016 33 Total phenolics Root (DCY) 12.8±0.12 mg/100g Faisal et al., 2016 34 Total phenolics Root (DCP) 54.62±1. 11 mg/100g Faisal et al., 2016 35 Total phenolics Root (DCW) 16.15±0.09 mg/100g Faisal et al., 2016

Phenolic acids like anthocyanins-compounds that consist of an antho-cyanidin in back-bone, 2-phenyl benzopyrylium also called as flavylium-cation. The generallysix anthocyanidin back-bones are pelargonidin, cyanidin, , delphinidin, petunidin peonidin and malvidin (Kammerer et al., 2003). Wu et al. (2006) described that In United state diet, every- day consumption of anthocyanins-compound is around 12.50mg/day. Invitro antioxidant capacity of purple carrots is greatly influenced by the presence of anthocyanins (Sun et al., 2009). Dietary anthocyanins may contribute to health-endorsement and defense from cardiovascular diseases (Reed, .2002; .Mazza, 2007) and degenerative diseases like tumor (Hou, 2003; Wang and Stoner, 2008) as they are innate anti-oxidants, decrease inflammation and lipid-oxidationand impart vaso-protective consequences. Anthocyanin compounds are

involved in improvingintelligence and memories (Shih et al., 2010) ”. In view of data regarding the phenolic acid contents determined by different techniques in a number of carrot cultivars from different regions of the world, it can be concluded that carrot is reach source of phenolic acids having high antioxidant activities. From these findings it may be suggested that production of carrots should be enhanced by using modern cultivation techniques to overcome the needs of antioxidant in the present era. Data related to phenolic acid may be helpful for the extraction of phenolic acids on commercial scales by process industries to produced anticancer and anti-aging medicines.

Where R: 2-xylose-6-galacto-side 2-xylose-6-sina-poyl-glucose-galacto-side 2-xylose-6-glucose-galacto-side 2-xylose-6-feru-loyl-glucose-galacto-side 2-xylose-6-(4-couma-royl)glucose-galacto-side Figure 2.2 The major phenolics acids present in D.carota is chloro-genic acid. Poly-phenols which are usually found in violet carrots incorporate 5-antho-cyanins. The fundamental antho-cyanin back-bone is known as cyani-din. The R-groups are singular glycol-sides.

2.6 Volatiles The compound psychoanalysis of vegetable volatile sec. metabolic-profile (e.g. reciprocal relation of primary and secondary complex) showed it dependence on sample’s groundwork process or diagnostic method working. It has been pointed out that during vapor cleansing, sample-composition might modify owing to the thermal deprivation of its principal compounds. (Rowan, 2011). The carrot is a popular vegetable, and its consumption is of high nutritional relevance. Carrot popularity has been attributed to its sugariness and pleasing taste, fulfilling munch and fitness reimbursement. The typical whiff and taste are mostly owing to its fickle-compounds (Alasalvar et al., 2001). Even though other compounds, like free sugars, non-volatile bitter compounds and liberated amino--acids have also been account to add to the sensory eminence. Mono-terpenes and sesqui-terpenes liable for on 98% of the sum of volatiles in carrot (Kreutzmann, et al., 2008; Simon, 1982). Soria et al. (2003) studied the volatiles compounds in dried out D.carota samples by solidphase micro--extraction tag along GC-MS. The G.C-M.S. chromatograms illustrate terpenoids pertinent to carrot smell such as alphapinene, sabi-nene, betamyrcene, limo-nene, gammaterpinene, terpino-lene, trans-caryo-phyllene and beta-bisabo-lene, and a number of furan imitatived. Fukuda et al. (2013) studied the aroma-characteristics and volatile-profiles of fourteencarrot varieties. The partial least-squares regression-analysis estimated the quantitative-contributions to insensitive and fruity carrot tang. It was experienced mono- terpenes had considerable positive-correlations with these attributes, while bisabolene isomers had negative correlations. The tangcharacteristicstrength &volatile contents and dietary-compounds are comparativelyless in Kuroda-type than in new carrot-types. They recommended that Kuroda is useful in reducing harshness of carrotsthroughout the growth of novelD. carota with good qualityeating. Kjeldsenet al. (2003) used headspace separation technique to collectvolatiles from the D. carota cut-strips. Thy characterized the volatiles by G.C-M.S, -FID, -M.S/M.S, and G.C- O. (Gas Chromatography - Olfactrometry) to hit upon t the volatile-formulation and tang- active compounds of D.carotaplacedinvariable temperature environments. Out of 52 measuredcompounds, mono- & sesqui-terpenes were dependableontotal volatile compounds. Core volatile constituents were beta-myrcene, (-)-alpha-pinene, (+)-limonene, (+)-sabinene,

p-cymene, (-)-limonene, terpinolene, gamma-terpinene, beta-caryo-phyllene, alpha-humu- lene, and (E)- and (Z)-gamma-bisabo-lene. A significant enlargement in the concentration of mono- and sesqui--terpenes was practical through chilled-storage and freezing-storage. G.C- O. exposed that the main volatiles jointly with (+)-alpha-pin-ene, (-)-beta-pin-ene, (+)-beta- pin-ene, 6-methyl-5-hepten-2-one, (-)-beta-bisabo-lene, beta-ion-one, and myris-ticin had an aroma feeling, which built-in annotations of "carrot-top", "terp-ene like", "sea green", "plain", "fruity", "citrus-like", "piquant", "timbered", and "sugary". The psychoanalysis of volatiles in dried out carrot section by solid-phase micro--extraction chased by G.C-M.S (Soria et al., 2008), showed that terpenoids contributing aroma to carrot were α-pin-ene, sabin-ene, β-myr-cene, limon-ene, γ-terpi-nene, terpino-lene, trans-caryophy-llene and β- bisabo-lene, and numerous furan imitatives. Kjeldsenet al. (2009) observed the studied momentous differences among the carrot verities regarding their constituents and volatile contents. They reported that carrot volatiles were complex mixture of terpenes, aldehydes, alcohols and sulfur and furanic compounds, which were considered to be responsible for flavour of processed carrot. Due to interactions between individual flavour compounds, even minor changes in the concentration of one compound may have a major impact on the overall flavour. Many compounds are responsible for carrot-flavor and a few of these might have impact on individual physi-ology. The characteristic fresh carrot-taste is generally due to the volatiles, mono-ter-penes and sesqui-ter-penes, and sugars also (Simon et al., 1980). Ter- penes contributed to mor-dant-taste and their taste-qualities were found to be directly related to the terpenes concentrations in diverse D.carota geno-types (Simon et al., 1980; Kreutzmann et al., 2008). The most abundant volatile compound seems to be terpinolene and the concentrations of volatiles fluctuates deeply between geno-types (Simon et al., 1980; Alasalvar et al., 1999, 2001; Habegger & Schnitzler, 2005; Kreutzmann et al., 2008). Violet carrots reported to contain comparatively low terpino-lene (Alasalvar et al., 2001) than wild cultivars. Catering procedure can reduce volatiles by 70-95% in carrot (Simon and Lindsay, 1983; Alasalvar et al., 1999). A cluster of amalgams called poly-acetylenes is accountable meant for the harsh- flavor of D. caroat (Czepa and Hofmann, 2004).There are four polyacetylenes presented in Figure 2.3 create in carrot core and the mainly plentiful are falcarindiol 3-acetate, falcarinol,

and falcarindiol, (Christensen and Brandt, 2006). Contents of poly-acetylenes in carrots range from 20-100mg/kg (Czepa and Hofmann, 2004; Zidorn et al., 2005; Christensen and Kreutzmann, 2007). Different cultivars have different concentration of polyacetylenes and it seems to be accumulated more in the carrot root phloem (Czepa and Hofmann 2004; Baranska et al., 2005). Carrots with privileged caro-tenoids had shown elevated concentration of poly-acety-lenes, and yellow, which have lower levels of carote-noids, had lower levels of polyacetylenes than orange carrots which have relatively higher level of carotenoid (Baranska et al., 2005). The falcarinol content was reduced by 70% as compared to rawcarrots once steaming carrots for 12min (Hansen et al., 2003). Many other compounds are responsible for distinctivearoma of carrots and people commonlyfound of carrots which have sweet taste and little volatile- terpenoid contents (Rubatzky et al., 1999). 2.7 Nutritional aspects and potential as functional foods Carrot is a cheap and important crop due to its diverse nutritional and health promoting properties, appealing many people from last few decades. Orange carrots are believed to have good effect on the eyes having high contents of caro-ten-oids, a set of phyto-chemi-cals that could easily converted to VitaminA in the body. Pre-formed dietary Vitamin A is obtained from animals and fortified foods or from plants as pro-vitamin-A carotenoids (Arscott & Tanumihardjo, 2010). D. carota do not give a notable-amount of calories in diet form, but accomplishto givevita- minted nutrition and phyto-chemicals, such as anthocyanins, carotenoids and other phenolic- acids. 100g serving of carrots provides more than 100% of daily value of vitamin A. Carrots also provide good daily values of vitamin K and vitamin B6 being 13% and 11% respectively (USDA, 2008). The maximum dietary attentions in carrots arise from their phyto-chemi-cal content, but carrots as a fiber resource have also been studied (Slavin & Lloyd, 2012). Carrot root is more or less 88.0% water, 1.0% protein, 9.0 % carbohydrate of which 5.90 % free sugars, 0.20% fat, and 2.30 % fiber (Table 2.5, 2.6). The carbohydrate component consists almost entirely of simple sugars, mainly sucrose, glucose, and fructose, starch is also present in small amount (USDA, 2008).

Figure 2.3 Structural formulas of terpinolene, falcarinol, falcarindiol, falcarindio 3- acetate

100g serving of raw carrot provides a good percentages of Recommended Dietary Allowance (RDA) to females elderly 19-30 years as: 120% vitamin--A (as RDA), 4.50% vitamin--E, 3.0%-calcium, 7.0%-potassium, and 11.0%-fiber (Arscott & Tanumihardjo, 2010). Dietary fiber is indigestible complex carbohydrates that are present in plant’s structural cells. Which cannot be engrossed by the human, therefore, give no energy however, the great health-attributes relate to eating of fiberrich food (Elleuch et al., 2011). Depending-upon-their-solubility fibers are of two types, insoluble and soluble. Cell wall components,typicallylignin and hemi-cellulose, cellulose, constitute insoluble fibers and soluble-fibers are noncellulosic poly-saccharides such as gums, pectin, mucilage (Yoon et al., 2005). The carrot wall is self-possessed of pec-tin, cellulose, lignin and hemi-cellulose (Lineback, 1999). High amount of dietary fiber is present in carrots (Bao & Chang, 1994), thus making it healthy food (Anderson et al., 1994). Foods abundant in dietaryfiber are accredited to the avoidance, lessening and handling of some illnesses such as cardiac diseases and diverticulosis (Gorinstein et al., 2001; Anderson et al., 1994; Villanueva-Suarez et al., 2003). The components of dietary fiber in carrot are 7.41 % of pectin, 9.14% of hemi- cellulose, 80.91% of cellulose and 2.48% of lignin (Nawirska &Kwaśniewska, 2005). 2.8 Pharmacological aspects/health benefits Carrot is not only useful as food but it can also be used in different medical fields. Carrot roots are utilized as refrigerant (Pant & Manandhar, 2007). They are used to cure kidney diseases, dropsy, nervine tonic and to repel uterine pain. Eating carrots boosts the quantity of urine. Considerable amount of carrots in diet and food has a positive impact on nitrogen balance (Anjum & Amjad, 2002). Carrot is consumed as fresh salad by astronauts in space missions (NASA Facts, 2002). Astronauts on space operations become more susceptible to high levels of waves, which endangers their health by putting them at lofty danger for a few kinds of cancers. The incorporation of carrot containing high level of beta-carotene in diet can decrease harmful effects of radiations and resist against cancer. In the process of curing chronic diseases, selecting and producing carrots with high beta-carotene values could be important (Kiremire et al., 2010). Besides that vitamin A deficiency (VAD) can be reduced by using high β-carotene carrots, especially in developing countries. The health of huge number of elders, adults and kids in developing countries is being affected by the global problem of VAD (Haskell et al., 2004). Goldberg et al., (1988) observed that people who ate

fruits and vegetables having high content of vitamin A more had suffered from macular degeneration way less than those who ate less. People having low carotene level in plasma are more vulnerable to death from ischemic heart disease (Stahelin et al., 1992). In view of data available about the health importance of carrot in several citations, it is very important to compile and categories the data in a sequence which is given below. 2.8.1 Antioxidant Potential An antioxidant is a molecule having ability to stop the chain reaction of oxidation by eliminating free radicals inter-mediates. Animals and Plants have a multifarious system of antioxidants to prevent this detrimental reaction (Singh et al., 2012). Extremely imprudent and destructive chain reaction of oxygen-species starts out as a result of oxidation process which is very destructive to biological systems in living organisms. The oxy-gen centered free-radicals and reactive-oxygen species (ROS) being constantly created cause the death of cells and tissues (Bailly et al., 2008). The damage caused by these oxidizing species is supposed to be responsible for the pathogenesis of numerous persistent diseases similar to cancer, diabetes neuro-degenerative diseases, athero-sclerosis, cirr-hosis, malaria and AIDS (Yagi, 1987; Yoshikawa et al., 1994; Reznick et al., 2006; Dias, 2012A). The major species involved in oxidative damage are superoxide-free-radical, hydroxyl-free radical, hydrogen peroxide and singlet-oxygen. As a consequence DNAmutation, protein in-acti-vation, rapid per-oxidation, and cell decease occurs (Vertuani et al., 2004). Anthocyanins were the main antioxidants pigments in purple-yellow and purple- orange carrots, and the main antioxidant found in all carrots was chlorogenic acid. Carotenoids did not affect the total antioxidant capacity, but showed a correlation with antioxidant capacity of hydrophobic extracts. Both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid (ABTS) tests proved that hydrophilic extract possessed greater antioxidant capacity than hydrophobic extract. The highest antioxidant capacity was manifested by Purple-yellow carrots, followed by purple- orange carrots (Sun et al., 2009, Faisal et al., 2016). According to ABTS and DPPH tests the purple–yellow carrots showed more than ninety period elevated antioxidant capacity than to the hydro-phobic mines.

Table 2.5 Proximate composition of carrots Proximates (%) Energy Reference Moisture Carbohydrate Protein Fat Fiber Ash Kcal/100g

88.29 9.58 0.93 0.24 2.8 nr 41.0 USDA, 2016

88 9.6 1.0 0.2 2.8 1.0 41.0 USDA, 2008 86 10.6 0.9 0.2 1.2 1.1 nr Gopalan et al., 1991 88.8 6.0 0.7 0.5 2.4 nr 30.1 Holland et al., 1991

86-89 6.6-7.7 0.8-1.1 nr nr nr nr Howard et al., 1962; Gill and Kataria, 1974

Table 2.6 Mineral composition of carrots Minerals (mg/100g) Reference Ca Fe Mg P K Na Zn

33 0.30 12 35 320 69 0.24 USDA, 2016 33 0.30 12 35 320 69 nr USDA, 2008 34 0.40 9 25 240 40 0.20 Holland et al., 1991 40.6 0.40 10.4 30.4 271 35.5 0.50 Krejčová et al., 2016 23 0.27 8 23 183 5 0.3 Decuypere, 2017

Table 2.8 Vitamin profile of carrots

Vitamins (mg/100g) Reference C B6 A E K B1 B2 B3 Niacin Folate, DFE

3985.8 10.7 0.051 0.034 0.503 2.80 0.119 0.8 mg - 11 µg Decuypere, 2017 µg µg mg mg mg

5.90 0.138 0.835 0.66 0.0132 0.066 0.058 0.983 nr 0.019 USDA, 2016

5.90 nr 0.835 0.66 nr 0.07 0.06 nr 0.98 Nr USDA, 2008

1.20-8.90 ------ERS, 2006

0.039-0.043 ------Howard et al., 1999

191-703 1.40-5.80 ------Nicolle et al., 2004 µg/100g

54-132mg/kg ------Matejková and Petriková, 2010

The higher antioxidant capacity values of purple carrots were owing to their antho-cyanin contents (Metzger & Barnes, 2009). The methanol extract of the aerial part of Daucus carota showed a stronger anti-oxidative activity than the standard synthetic antioxidant, 3-tert-butyl- 4-hydroxyanisole (BHA) using the ferric thiocyanate method (Ono et al., 2001). The antioxidant capacity of raw and processed purple D. carota was measured by DPPH method showed the EC-50 value varied from 7.80-30.23mg/100g (Uyan et al., 2004). Flavonoids and phenolics found in carrots also play an important role as antioxidants. Chloro-genic-acid, the main hydro-xyl-cinnamic-acid, ranged from 42.20 to 61.80% of total phenolics investigated, is reported to be involved in some strong antioxidant activities as well. The ability of all phenolic extracts to absorb free radicals was stronger than pure chlorogenic acid, vitamin C and β-carotene (Zhang &Hamauzuet, 2004).

2.8.2 Anti-Inflammatory Potential The experimental work confirmed anti-inflammatory effects of carrot seed extract. Anti- inflammatory positional was due to the presence of 2,4,5-trimethoxybenzaldehyde, oleic acid, trans-asarone and geraniol, which obstruct the cyclo-oxygenase enzymes. The anti- inflammatory benefits of these compounds were comparable to synthetic drugs like Aspirin, Ibuprofen, Naproxen and Celebrex (Momin et al., 2003). In a study, carrageen an induced rat paw oedema was cured from 60 to 70% by volatile oil of carrot seeds at the dosage of 50 to 100 mg/kg body weight (Prochezhian & Ansari, 2000). Vasudevan et al. (2006) reported that carragee-nan-, hist-amine- and seroto- nin- provoked oedema as well as form-aldehyde-stimulated arth-ritis in rats were considerably inhibited by extract of seeds when administered at the pace of 200 and 400 mg/kg. Lipo-polysaccharide (LPS) inflammatory response was weakened by a bioactive – chromato-graphic part of purple D. carota extort. Nitric oxide production and mRNA of pro- inflammatory cytokines were also reduced, depending upon the dose administrated and Inducible nitric oxide synthase (iNOS) in macrophage cells. The production of nitric oxide was decreased up to 65% in macrophage cells without cytotoxicity due to the action of falcarinol , olyacetylene falcarindiol and falcarindiol 3-acetate (Metzger et al. 2008 ; Metzger & Barnes 2009).

Inflammatory bowel disease (IBD) is a chronic disease whose cause is not yet known, results in chronic and spontaneously relapsing inflammation (DuPont & DuPont, 2011). It was observed that acetic acid induced experimental colitis in wistar rats was influenced by aqueous extract of Daucus carota (AEDC). In that study the animals were pretreated with Daucus carota (100, 200 and 400 mg/kg, p.o.) for 7 days before the colitis was induced in them. Stool consistency, colon weight, colon width, colon weight to length ratio, spleen weight, ulcer area, ulcer index, colonic MPO and nitric oxide was notably reduced by the pretreatment with Daucus carota aqueous extract (200 and 400 mg/kg, p.o.) for 7 days (Patil et al., 2012a). In another research aqueous and methanolic extracts of Daucus carota (DCAE & DCME) showed highest anti-inflammatory activity when administered at the rate of 400 and 140 mg/kg body weight with 90.9 and 58.6 % inhibition respectively on acute inflammation. The maximum anti-inflammatory activity was shown by the same dose with 58 and 44.1 % inhibition respectively, in chronic inflammation. DCME cured ethanol induced gastric ulcer with a ratio of 46.8 and 68.7% respectively, at a dose of 250 mg/kg body weight. Conclusively, both DCAE and DCME demonstrated auspicious anti-inflammatory potentials while showing no negative effects on liver, kidney and pancreas function (Wehbe et al., 2009).

2.8.3 Anti-cancer potential There are plenty of evidences available which suggests that the consumption of vegetables and fruit rich in antioxidant compounds decrease the risk of cancers. Carrot is rich in some beneficial agents like β-carotene and polyacetylenes, which could be helpful in the treatment of leukemia. A study was conducted by Zaini et al., (2011) to examine how the myeloid and lymphoid leukemia cell lines as well as normal hematopoietic stem cells are affected by carrot juice extracts. Leukemia cell lines and nontumor control cells were treated with carrot juice extracts for up to 72 hours in vitro. The initiation of apoptosis was analysed by means of annexin V/propidium iodide staining following the flow cytometric analysis, and results were confirmed by using 4-6- diamidino-2-phenylindole morphology (Yu et al., 2007). Cell cycle analysis and cell counts were utilized to investigate the effects on cellular proliferation. Carrot juice extract induced apoptosis and prevented the progression through the cell cycle.

Lymphoid cell lines were influenced more than were myeloid cell lines and normal hematopoietic stem cells got least affected. That research had indicated that apoptosis and cell cycle arrest in leukemia cell lines can be achieved by using extracts from carrots. It was concluded from these findings that carrots may prove to be a treasure of bioactive chemicals for the treatment of leukemia (Zaini et al., 2011). New therapy techniques for the treatment of leukemia are in urgent need to be discovered. In traditional medicine the carrots have been considered to possess a significant potential for the cure of leukemia and previous researches in other contexts have reported carrots as excellent source of anticancer agents. In a study the impacts of five fractions from Carrot Juice extract (CJE) on human lymphoid leukemia cell lines were studied and found that in selected carrots, polyacetylenes were the bioactive components rather than beta- carotene or lutein and could be beneficial in the development of new leukemic therapies. Induction of apoptosis and arrest of cell cycle had been observed under the cytotoxic effects of polyacetylenes for the first time in that study (Zaini et al., 2012). On the other hands it was observed that normal and cancer cell proliferation were inhibited notably by fractions of falcarinol, falcarindiol, and falcarindiol 3-acetate from carrot extract (Purup et al., 2009). The study reveals that the aliphatic C17-polyacetylenes were the prospective anti-cancer agents of carrots and co-operative interactions between bioactive poly-acetylenes may be important for their bioactivity. Anti-tumour activity, preventing the development of Ehrlich ascites tumour in mice was observed when the gasoline-ether extort of carrot-kernel was taken to them intra- peritone-ally at doses of 03.0mg and 010mg/kg of body wt/day for week (Majumder & Gupta, 1998). The progress of large abnormal crypt-foci & azoxy-methane AOMinduced colon preneo-plastic tumors in male-BDIX mice was held up when usual rat-feed Altromin was treated with either D. carota containing 35.0μg/g falcari-nol or corn starch augmentedwith 35.0μgfalcari-nol/g extracted from carrot (Kobæk-Larsen et al., 2005).Significant antitumour activity was illustrated by carrot umbel oil extract beside 7, 12- di-methyl benz (a) anthrax-cene DMBA-induced-skin-cancer in opposite to non - care for rats. It was observed that carrot oil retarded tumour appearance (Zeinab et al., 2011). It was studied that phloretin and quercetin, secluded from carrot-roots, were efficient in reducing the possibility of human-colon melanoma (HT29 and LoVo cells). The possibility of colon

cancer was remarkably brought down by the addition of chlorogenic acid and caff-eic-acid, found in carrots. Caffe-ic-acid was notably more efficient, diminishing endurance of HT-29- cell to 62.060% and 25.070% at 500.0μmol/L & 1000μmol/L, correspondingly. Yet caffeic- acid affected the LoVo cells less than it did-HT29 cells and their possibility was reduced to 68.27 % after 24hours at the highest concentration 1000 μmol/L(Przybylska et al., 2007). In view of data regarding anticancer properties of carrot extracts on animals, it may be concluded that carrots cultivars have much potential against different types of cancers and can be used in the preparation of anticancer medicines at commercial scales, not only for animals but also for human beings.

2.8.4 Protective potential for cardiovascular diseases Carrots also reduce the chances of heart attack in women (Gramenzi et al., 1990). In a study conducted by Griep et al. (2011), the relationship between the fruits and vegetables of various colors and 10-year coronary heart disease (CHD) incidence was observed. The data was based on a prospective population-based cohort having 20,069 men and women aged 20 - 65 years, recruited between 1993 and 1997. It was found that the consumption of more dark-orange-colored vegetables and fruits leads to a lower the threat of CHD. Particularly carrots, which contributed most to their total orange fruit and vegetables consumption being 60 %, were associated with a 32% lesser risk of CHD. It was deduced that greater intake of deep orange fruit and vegetables, and especially carrots, might provide protection against coronary heart disease (Hertog et al., 1995). The hazardous impact of isoproterenol-induced heart attack in rats was improved by administration of carrot extract (Muralidharan et al., 2008). Serum aspirate trans-aminase and ala-nine trans-aminase, lact-ate de-hydro-genase, fat per-oxi-dase levels were decreased by Isoproterenol and carrot extract reduced lofty lipid per-oxi-dation in heart-homo-genate. D. carota extort restored the lower whole-protein and lact-ate-de-hydro-genase echelons in the heart-homo-genate close to standard stage (Muralidharan et al., 2008). Adultsmay protect the cardiovascular system by drinking carrot juice which result in enhanced total antioxidant status and lower lipid peroxidation irrespective of any of the cardiovascular risk markers measured. The levels of plasma cholesterol, triglycerides, Apo A, Apo B, LDL, HDL, body fat percentage, insulin, leptin, interleukin-1a or C-reactive protein were not influenced by

carrot juice. Drinking carrot juice decreased systolic pressure, but did not have any impact on dia-stolic-pressure. Consuming D. carota fluid notably improved the blood total-anti-oxidant activity (Potter et al., 2011). From the review data related to protective potential of carrot against heart diseases it may be concluded that raw carrot can be used not only as a remedy but also for protective sheet against cardiovascular diseases induced due to various factors.

2.8.5 Hepatoprotective, wound healing, anti-bacterial and antiviral activities

The impact of carrot extract on carbon tetrachloride (CCl4)-induced severe liver damage was assessed in a study conducted by (Bishayee et al.,1995). CCl4-induction raised serum-enzyme echelon (viz., gluta-mate oxalo-acetate trans-aminase, gluta-mate pyru-vate trans-aminase, lact-ate de-hydro-genase, alkaline-phospha-tase, sor-bitol and glut-amate de-hydro-genase) and pretreatment with the extract showed significant declined. The boosted serum bilirubin and urea content, due to CC14 administration, were also lowered by the extract. CC14 boosted the activities of hepatic 5'-nucleotidase, acid phosphatase, acid ribonuclease and lowered the levels of succinic dehydrogenase, glucose-6-phosphatase and cytochrome P-450 but this effect was reversed by the extract in a dose-responsive way. It was divulged from the results of this study that carrot could relieve CCl4-induced hepato-cellu-lar-in-jury significantly (Bishayee et al., 1995). Bello et al. (2012) studied the effects of pretreatment and administration of aqueous extract of Daucus carota root on hepatotoxicity in rats. It has been found that Anti-tubercular drugs caused hepatotoxicity in patients who were under the treatment of tuberculosis. The outcomes of this study indicated that aqueous extract of D. carota root enclosed considerable hepato-protective potential, which proved the fact that I could be a new drug for the inhibition of liver injuries on the other hands Singh et al., (2012) investigated the hepatoprotective effects of methanolic extracts of D. carota seeds on thioacetamide-induced oxidative stress in rats liver .They found that treated rates urine contained significant lowered levels of liver enzymes and concluded that D. Carota seed extract had contributed in the fortification of liver in rats. From the findings of published citations related to hepatoprotective activity of carrot extracts in animals showed that carrot has much potential

for the remedy of liver diseases and can be used for the extraction of hepatoprotective agents to produce liver protective medicines on commercial scales. Patil et al. (2012b) prepared three formulations ((1 %, 2 % and 4 % w/w)) by using ethanolic extract of carrot in the form of cream. They applied this formulation on wounds and found notable increase in protein and hydroxyproline contents (Figure 2.4) causing no irritation

Figure 2.4 Photographs of rats showing different stages of wound healing (Patil et al., 2012b). A: Vehicle Control B: EEDC (2% w/w) C: EEDC (4% w/w) Rossi et al., (2007) reported that the activity of the enteropathogen Campylobacter jejuni was blocked by the essential oil extracted from top whole parts of wild carrot. Compounds extracted from essential oil like phenylpropanoids, such as methylisoeugenol and elemicin showed antimicrobial activity against Campylobacter coli and C. lari strains. In another cited research the anti-bacterial activity of methanolic extract of carrot seeds was examined. The main flavones extracted by using methanol were Luteolin, luteolin 3'-O-β-D- glucopyranoside, and luteolin 4'-O-β-glucopyranoside. It was observed that luteolin showed anti-bacterial activity against Bacillus cereus and Citrobacter freundii. B. cereus and Lactobacillus plantarum were inhibited by luteolin 3'-O-glucoside. Likewise luteolin and 4'-

O-glucoside hampered the growth of Staphylococcus aureus and Escherichia col (Kumarasamy et al., 2005). The antiviral activity of polyphenols, extracted from foliage and heredity of carrot was also explored. It has been observed that herpes simplex virus type 1 (HSV-1), a recurrent human virus with no medical cure. The plaque diminution assay was used to investigate the antiviral activity of extracts against HSV-1. Variations in virucidal activity were revealed by direct contact between the HSV-1 and the extracts in cell-free assay system, depending on the polyphenolic contents of carrot extracts. To investigate other practicable mode of actions, vero cells were treated with extracts before, during, and after viral infection to recognize the impact on virus life cycle. The HSV-1 viral replications were inhibited by the application of extracts, resulted in significant anti-viral activity (Torky, 2013). Carrot has significant nutritional and health attributes which govern to include carrot in human and animal diet. Carrots are rich in phenolics, carotenoids, vitamins and minerals which helped to reduce the risk of some harmful diseases. Researches in several studies concluded that carrots have anti-carcinogenic, anti-oxidative and immune-enhancer effects. The review of precedent studies has been discussed in this section which revealed that physic-chemical attributes of carrot have gained much gratitude among food researchers and scientists because of its multifold functional properties. Although, a large numeraldata about D. carota cultivars have been cited regarding functional and biological properties.Conversely, to the best of our knowledge there is no previoustestimony yet publishedhavingcomprehensive detail about chemical-characterization and assessment of biological-activities of novel cultivars of D. carota, native to Pakistan

Chapter 3 MATERIALS AND METHODS The presented research work in this dissertation was performed in the Laboratories of the Department of “Applied Chemistry and Biochemistry”, Government College University Faisalabad, Pakistan, University of Veterinary and Animal Sciences, Lahore, Pakistan and Prime Care Clinical Computerized Lab., Faisalabad, Pakistan,

3.1 Materials

All reagents and chemicals utilized in this research were of ”analytical grade” purchased from . Merck, Sigma and Fluka. ”1, 1-diphenyl-2-picry.lhydrazyl radical (DPPH ) (.Sigma, 90.0 %)”,

”lin. oleic-acid ”, β -. carotene”, Folin. -Ciocalteu ” reagent (2N) ”, All other chemicals (analytical

grade) i.e. ”anhydrous. sodium-carbonate.”, ” sodium-hydroxide ”, ” sodium-nitrite ”, ” ferrous chloride ”,

”ammonium-thiocyanate”, ”aluminum-chloride”, ” potassium-dihydrogen” -phosphate”, ” di-potassium

hydrogen” -phosphate ”, iso-octane” , chlor.oform” , acetic” acid, potassium” -iodide, and ” sodium-

thiosulphate” used in this study were ” purchased from .Merck ” (Darmstadt, ” Germany),

otherwise”.specified”.

3.1.2 Standard compounds

All the standard chemicals used in this research were of analytical grade i.e. ”Standards of

”phenolic acids ‘(3,4--”” diferuloyl-quinic-acerbic, ” cis-3-‘ -” caffeoyl-quinic-”acid, 5--‘ ”caffeoyl-

quinic-acid, Caffe-ic ” -acid, 3,5--”” dicaffeoyl-quinic-acid, 3 ” -p—coumaroyl-quinic ” -acid, 3 ”—

feruloy-quinic” -acid, 3,”4--” dicaffeoyl-quinic-acid, ”5 ” --feruloy-quinic-acid, 3--” caffeoyl-quinic-

acid,, ” cis-5--” caffeoyl-quinic-acid, ”5--p-”coumaroyl-quinic-acid, ”4--” feruloy-quinic-acid, and 3,

”5--” diferuloyl-quinic-acid) and standards of sugars (maltose ”, maltotriose”, maltodextrin”,

glucose” and fructose”) were purchased from ”Sigma Chemicals Co (St, ”” Louis, ”MO, ” USA)”.

3.1.3Instruments Table 3.1 Description of instrumentsDescription used throughout the research work

Instrument Name Model Manufacturing company

HPLC-RID LC-20A Shimadzu, Japan

HPLC-L-4500 Diode array L-4500 Hitachi Ltd., Tokyo, Japan detector Hitachi Instruments Inc. Tokyo, Spectrophotometer U-2001 Japan

Digital Refractometer HI96800 HANNA ,United Kingdom

Commercial blender TSK-949 Westpoint, France

Hot air oven IM-30 Irmeco, Germany

Orbital shaker UNIMAX 2010 Heidloph, Germany

Kjeldahl apparatus Labconco-2123205-040520113 Labconco (USA)

Soxhlet apparatus LABGO 501 LABGO 501(India)

Huanghua Guangming Magnetic Stirrer SH-2 Instrument Co., Ltd.China

Electric Balance MP-300 Ohyo, Japan

Centrifuge H-200NR H-200NR Kokusan, Japan

Rikakikai Co. Ltd. Tokyo, Rotary vacuum evaporator EYELA, N-N Series Japan

3.1.4 Collection of samples The selection of carrots wasmadeby considering the related informations and intensive reviews. For expediency the carrots were categorized on the basis of cultivar nature. Root part and top whole of seven approved cultivars DCP,DCW, DCY, DC90, T29, DCR and DC3 and were collected in the month of January 2015 and seeds in the month of October 2014 from the fields of vegetable-section Ayub-Agriculture Research-Institute Faisalabad Pakistan. Cultivation of understudied cultivars of D. carota was completed in winter season (October-2014 to January-2015). Samples were collected according to the plan of study in the month of January. Table 3.1 presented thecomprehensive data regarding temperature (minimum/maximum), relation wetness (average) and rainfall(total) for months wise of cultivation The samples were further authenticated and verified by Dr. Qasim Ali, Assistant Professor of Botany, Government College University Faisalabad. 3.1.5 Preparation of the plant material for chemical analysis The carrot-root and top-whole samples of all cultivars were sponged down with tap-water followed by deionized water. Then samples were dried with paper-towel, then residual moisture was removed by drying at room temperature, then drying was done by placing samples at 55.0ºC for twenty four hours (Abuyeet al., 2003). The dehydrated carrots and top whole of individual cultivar were grinded to obtain powdered samples separately using pestle-mortar, and sieved by using sieve of 20-mesh. The seeds of respective cultivars (without pretreatment steps) were also ground into powder separately using pestle and mortar, and sieved through 20-mesh sieve. The dried powder samples were used for further analysis. All the prepared-samples were raped in cleaned-polythene bags and stored in refrigerator at -10ºC.

3.3 Experimental protocol Part 1 3.3.1 Proximate analysis of carrot cultivars The methods recommended by the Association of Official Analytical Chemists (AOAC) were used to determine ash (#942.05), crude lipid (#920.39), crude fiber (#962.09) and nitrogen content by method #984.13 (AOAC, 1990)

Table 3.2 Description of the carrot roots and top whole employed in the current study Cultivar Maximum Minimum Relative Part used Cultivation Month Location Total rainfall selected Temp (°C).e Temp (°C). humidity October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DCW Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01 October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DCY Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01 October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DCP Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01

October 2014 35.6 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DCR Root and December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10 Top whole January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01

Continued Table 3.1 October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DC3 Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01 October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DC90 Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01 October 2014 35.7 ± 3.2 20.2 ± 3.0 48.0 ± 9.5 1.26 Root and November 2014 Fields of 25.5 ± 1.9 14.2 ± 2.4 68.0 ± 14.4 2.74 DCW Top whole December 2014 AARI 21.4 ± 2.3 8.3 ± 1.1 79.0 ± 18.5 8.10

January 2015 19.3± 1.5 8.0 ± 1.2 67.0± 13.6 0.01 AARI, Ayub Agriculture Research Institute Faisalabad Pakistan (Timeanddate, 2017)

a) Moisture content Moisture content of selected cultivars of carrot root, top whole and seeds was investigated by using oven (hot air). Drying of aluminum dishes was done at 105°C for 6.0 hours and then dried dishes were placed in desiccators, cooled to ambient temperature (25°C). 10 gram of greaten carrot weighed accurately in a clean, pre-weight aluminum dish by means of an analytical weighing-balance. Drying of samples was done at 70°C using hot-air oven for 18-22 hours unless steady-weight attained. Cooling of samples after drying was done in desiccators containing silica gel and re-weighed for calculation of moisture contents. Moisture analysis of all the samples was done in triplicate. Moisture contents of respective samples were calculated by means of the subsequent formula (AOAC, 1999). The results were expressed in g/100g.

Initial weight − Oven dry weight Moisture % = x 100 Oven dryweight b) Dry matter determination. In view of unstablemagnitude of moisture contents in selected cultivars, all results were calculated made on dry-matter basis. Dry-matter was determined by means of AOAC procedure (925.10) by using following formula.

Dry Matter % = 100 − Moisture % c) Ash content Ash content of selected cultivars of carrot was determined by the international organization method (AOAC, 1990) by using muffle furnace. Briefly, 2.0 g ground material of selected cultivar was precisely weighed in a pre-weighted crucible by means of an analytical balance and carbonization was done by heating on electric-flame. After carbonization samples were shifted to muffle-furnace and ignited at 550°C until constant weight attained.

weight of ash (g) Ash % = X 100 weight of sample (g)

d) Determination of crude fat and fiber contents For the determination of crude fat contents in carrot cultivars AOAC (1990) method was used. Briefly, 2.0 grams of sample from respective cultivars were placed in porous thimble of soxhlet apparatus and plugged it with mounted cotton wool. Porous thimble was placed in extraction chamber which was fitted on receiving flask (pre-weight) having petroleum eather (b.p. 40 to 60ºC). the extraction of crude fat was done by heating the mantle for eight hours. After described time the thimble was separated from soxhlet apparatus by distilling off the solvent. For removing residual solvent contents from oil flask containing fat contents was placed in oven at 100ºC for one hour. After cooling in desiccators, the flask was re-weighed for the determination of crude fat in the respective samples , and reweighed. The difference in weight was expressed as percentage crude fat content. Acid and base digestion procedure was used for the dtermination of crude fiber i.e. 1.25% solution of analytical grade sulphuric acid and 1.25% solution of sodium hydroxide were used to digest the samples separately. The residue (after extraction of crude fat process) was put into boling 1.25% solution of sulphuric acid in 600mL beaker. This solution was boiled for half an hour. Filtration was done through filter paper after cooling the residue. Then washing of this residue was done by using 50ml bloimg water in three portions. After complete washing from sulphuric acid, the residue was put back to the 600mL beaker containg 200mL of 1.25% sodium hydroxide solution. This solution was boiled for half hour to complete the digestion process. To obtain washed residue, the washing was done by boiling water for three times. Final washing was done by using

25mL of C2H5OH. Then drying of residue was done at 130ºC till constant weight was attained, cooled in silica gel containing desiccators. Residue was transferred to pre- weighed crucible (porcelain), ashed at 550ºC for 2-3 hours, cooled and re-weighed. The calculations were done by means of following formula. Crude-fiber was represented % loss in weight on-ignition

weight of loss on ignition (g) Crude fiber (%) = X 100 weight of sample

Loss on ignition (g) = residue (g) – ash (g)

e) Determination of nitrogen content and estimation of crude protein for the investigation of nitrogen contents in samples of respective cultivars , Kjeldahl- method was applied. Briefly, two grams of dried material was digested with 15mL of concentrated H2SO4 in kjeldahl flask using digestion tablets as catalyst, heating was continued until the clear solution was obtained. The digested solution was cooled and made the volume upto 400-500mL with distilled water. A pich of Zn-dust was added in diluted solution followed by 40.0mL of 50.0% NaOH solution. Boiling of this mixture resulted in the formation of ammonia, which was collected in receiving flask on 500mL capacity having 35mL of 0.01N solution of H2SO4 using methyl red as an indicator. Amount of ammonia was calculated by titrating the receiving flask solution with 0.01N of NaOh solution. Blank was also determined in similar way without using the sample material. Protein contents were estimated by multiplying the nitrogen contents with 6.25, a factor used to estimate the crude protein contents in material having nitrogen cintents (AOAC 1990). The nitrogen and protein contents were determined by means following formulae.

volume of acid used × dilution volume × 0.0014 Nitrogen % = × 100 Sample weight × diluted solution used (mL)

Crude protein % = Nitrogen % × 6.25 f) Estimation of carbohydrates and energy values Carbohydrates contents were determined by subtracting the total percentage of crude-fat, -protein and ash from 100% dry-weight of the sample material. The calorific energy (in KJ) of sampling material was determined by multiply the %age of crude-protein, crude- fat, and carbohydrates by 16.70, 37.70, 16.70 respectively (AOAC, 1990) g) Mineral analysis by atomic absorption spectrophotometer 10.0 grams of dried sample material of each cultivar was weighed in digestion flask followed by 20.0mL of 14.4mol/L HNO3 solution, 10.0mL of 30percent H2O2 solution and 10.0mL of 9.90 of perchloric-acid. Then 2mL of 18mol/L of H2SO4 solution was added to avoid losses of metal-halides through valtyilization (Feinberg & Ducauze, 1980; Erwin & Ivo, 1992). All the digestion flasks were reflux at 170°C for three hours to obtain clear digested solution (Yaman & Gucer, 1995). Centrifugation of digested

material was done to separate the clear solution and residue material was washed by using doubled-distilled water and re-centrifugation results in the prevention of elemental-losses. The first washing from the residue was added to original solution before distillation. The analysis was done by using stock solution. The amount of Co, Cu, Zn, Fe and Sr was investigated through atomic absorption spectrophotometer technique in “Unicam 939” apparatus. 3.3.2Estimation of physico-chemical properties of carrot seed oil a) Acidity Acidity is defined as the un-combined fatty acids present or formed in fats and oils due to the presence of moisture contents, high temperature and deterioration processAcidity due to free fatty acids is the KOH-milligrams used for neutralization of 1.0 gram of oil or fat sample. Amount of acidity was determined by following the previously cited method with slight-modification (Anwer et al., 2007). Two grams of oil sample were weighed in

100mL glass beaker, poured 25ml of pure C2H5OH in boiling form in the oil sample and placed it on hot plate. Few drops of phenolphthalein were added as indicator. Agitation was done by using magnetic stirrer to ensure the proper mixing of the sample. The oil sample was titrated (in the boil form) against 0.05N sodium hydroxide solution till the appetence of pink colour (persist for 30seconds).The acidity as oleic acid was calculated by means of following formula;

” NaOH (mL) X NaOH Normality X 282 Acidity as oleic acid (%)= sample weight

b) Peroxide value Peroxide value is used to determine the peroxides present or formed due to oxidation of lipids which oxidizes the potassium iodide. For the determination of peroxides in the oil samples of respective cultivars AOAC method # 965.33 was used (AOAC, 1980). Briefly 1.0g sample was taken in 100ml flat bottom flask, then 30.0mL of acetic-acid/ chloroform mixture (2:3) and 1.0g of potassium iodide (saturated) was added. Shacked for 60 seconds and 50mL of double distilled-water was poured in the sample flask. Mixture in the flask

was titrated against 0.01M sodium thiosulphate solution, vigorous shacking of solution in flask results in the disappearance of yellow colour. 0.3mL of starch indicator (1%) was added and continued the titration until the disappearance of blue colour. After knowing the used volume of sodium thiosulphate solution following formula was used for the estimation of peroxide value.

Volume of sodium thiosulphate used ×normality of sodium thiosulphate ×1000 PV= Sample weight

= mili − equivalents of hydrogen peroxide per kilogram of oil c) Saponification value

Saponification of 1.0 gram of oil or fat done by milligrams number of KOH is called as saponification value (SV). Standard IUPAC method was used for the determination of SV (IUPAC, 1979). Briefly, two grams of oil/fat sample was weighed in round bottom flask (250mL) followed by 0.5N KOH (alcoholic solution), refluxed the flask contents fo half hour, cooled and titrated against HCl (0.1N solution). Simultaneously blank was also determined in the similar conditions simultaneously.SV was calculated by means of following formula.

{ blank (mL)used − sample (mL)used}x28.05 SV = sample (oil)weight

“

D) Un-saponifiable matter 1.0 gram of oil sample from respective cultivar was dissolved in 0.5 N KOH (alcoholic) solutions in 250mL flat bottom flask. The flask was connected to condenser fitted with digital water bath system, boiling of sample at water bath results in the saponification of oil. Shaking and refluxing was performed for 6 hours to ensure the reaction. The saponied material from the flask was transferred to separating-funnel and washing was done with distilled-water. 50.0 mL dimethyle-ether was used for rinsing, rinsed dimethyl ether was poured in the separating funnel also. Shacked the funnel vigorously for 2 minutes and two lyres were separated clearly after 3 minutes settling time. The organic layer (ether) on upper side contained un-saponifiable matter, separated in 200ml beaker and washing with

distilled water was done until non alkaline water is obtained. The ether contents were removed by heating the residue at the 80OC for 30 minutes. The dried reside was cooled in desiccators and un-saponifiable material was weighed. The caluctions were done by using following formula. AOAC # 933.08 (AOAC, 1980)

“

weight of unsaponifiable matter Unsaponifiable matter (%) = × 100 Weight of oil

e) Specific gravity Specific gravity was measured at ambient temperature (25Ċ) by using pycnometer of 10mL capacity Briefly, pycnometer was dried to remove any moisture contents and placed it in desiccators, cooled to ambient temperature (25Ċ). Weight of empty pycnometer with stopper (W) was noted. Filled the oil up to the mark, close the mouth with stopper and noted the weight (W1). Same procedure was performed with distilled water by using the same pycnometer and noted the weight (W2). Calculations were made by using the following formula. Method # 920.140 (AOAC, 1998).

W1 − W 푆pecific gravity = W2 − W

f) Refractive index Refractive index of oil samples of respective cultivars was investigated by AOAC # 921.08 (AOAC, 1980) using a digital refractometer. Briefly, the mode “refractive index with temperature” was selected by clicking button “Range” applied the oil sample on sample well, closed the lid and clicked on “read” button, noted the reading. All samples were analyzed separately after washing the sample well with ethanol to remove previous sample contamination.

3.3.3 Estimation of physico-chemical properties of D. carota juice Extraction of D. carota juice Carrots from each cultivar were topped, tailed, washed thoroughly and scrapped before extraction of juice. The extraction of juice was done by Juicer machine, Philips (model # HL7715/00, 700-Watt). a) pH pH of the juice was measured by previously established method (Garner et al., 2005). Briefly, juice was filtered through cheesecloth. pH of the D. carota juice was determined at room temperature (25Ċ) by simple dipping the electrode of pH meter into filtered juice and recorded the values. b) Acidity Acidity of the juice was measured by previously established method (Vandresen et al., 2009). Briefly, 100mL juice was filtered through cheesecloth and titrated with 0.1N NaOH solution until pH value of 8.20 was attained. Acidity was calculated by using the following formula and reported as mg/100mL of juice.

Titer mLs − 0.1N NaOH 퐴푐푖푑푖푡푦 (푚푔/100푚퐿) = mLs of juice b) Total soluble Total soluble were determined by previously established method (AOAC, 2000). Briefly, mixed the juice thoroughly, filtered through filter paper (what No1) and transferred 50mL of filtrate to pre-weight evaporating dish. Evaporation was done at 100Ċ till to dryness under vacuum. Total soluble were calculated by the following formula and reported as % (W/V).

Residue weight(g) 푇표푡푎푙푆표푙푢푏푙푒(%) = X100 mLs of juice c) Total solids Total solids were determined by previously established method (AOAC, 2000). Briefly, mixed the juice thoroughly, and transferred 50mL to pre-weight evaporating dish. Evaporation was done at 100Ċ till to dryness under vacuum. Total solids were calculated by the following formula and reported as % (W/V).

Residue weight(g) 푇표푡푎푙 푠표푙푖푑푠 (%) = X100 mLs of juice d) Refractive index Refractive index of juice of respective cultivars was investigated by AOAC # 921.08 (AOAC, 1980) using a digital refractometer. Briefly, the mode “refractive index with temperature” was selected by clicking button “Range” applied thoroughly mixed juice on sample well, closed the lid and clicked on “read” button, noted the reading. All samples were analyzed separately after washing the sample well with distilled water to remove previous sample contamination. d) Brix Refractive index of juice of respective cultivars was investigated by AOAC # 932.14 (AOAC, 1980) using a digital refractometer. Briefly, the mode “Brix%” was selected by clicking button “Range” applied thoroughly mixed juice on sample well, closed the lid and clicked on “read” button, noted the reading. All samples were analyzed separately after washing the sample well with distilled water to remove previous sample contamination.

Part II 3.4 Chromatographic analysis 3.4.1Phenolic acid profile by HPLC 3.4.1.1 Sample preparation for the determination of phenolic compounds by HPLC Phenolic acids were extorted and decontaminated/purified following the previously established methods (Mayen et al., 1997) with slight adjustments. Blending to homogenization of 100g grated-carrotswas done in 100mL of 80 % cold-ethanol with sodium-metabisulfite (0.5%) and left it for half hour and repetition (3times) of same experiment was don, alternately. Cheese-cloth was used for the filtration of prepared material.By using 50mL of same solvent 3 successive-extractions were done repeatedly. Centrifugation of filtrate was done for fifteen minutes at 7000 rpm. Evaporation of

C2H5OH was done in vacuum at 35 to 40°C. Removal of colorants was done by means of

petroleum.20.0 % of ammonium-sulfate and 2.0 % of metaphosphoric-acid was mixed with aqueous-phase,and then ethyl-acetate was added in the ration of 1:1 for the extraction of phenolic compounds. Evaporation of ethyl-acetate was done at 35 to 40°C to get the dried material. Finally, 10.0mL of CH3OH was mixed with shaking to obtaine cleared and homogenized solution of the extract and stored at-20 °C. this prepared sample was further used for the analysis of phenolic compounds by HPLC. 3.4.1.2 Exploration of phenolic compounds by HPLC The phenolic acids were investigated on qualitative and quantitative bases by method of

HPLC-D system having pump. -L-62.00-A, di-ode-array-de-tector (L-4 .500-Hit.achi Ltd---

Tokyo--Japan), and PC-Elonex. ---Pc--466./I (Elone. x—As-pl. ey---method,-London--U.. K.). Syringe filter of 0.45µm was used for the filtration of samples to get micro-free sample. For the gradient separation of phenolic compounds Alltima-C18 column having 250mm,

4.60mm int.D was used.’ All-tech-connections dev. Science Ltd., ’Carnforth ’, U.K’) was practiced for gradient partition of the phenolic-acid. Mobile--phase made of A-solvent

(2.0% aceticacid) ’and B-’ solvent methanol-/-acetonitrile’ (15.0:10.0-v/v). Flow speed of 1.00mL/min was achievedby using pump of above specifications. Following gradient elution was used for the complete exploration and separation of phenolic compounds: at 0min, (A--90%) and (B--10%) ;@10.0min, (A--80%) and (B—20.0%); @15.0min, (A-- 70%) and (B--30%) ;@25min, (A—60.0%) and (B—40.0%); @30min, (A-50.0%) and (B-50.0%) and @40min, (A--50.0%) and (B—50.0%) . By setting di-ode-array-de-tector

at 330nm was used for examination and partition of phenolics (Alasalvar et al., 2001).’ 3.4.2 Saccharides (sugars) profile by HPLC a) Sample preparation for sugars analysis Toping and tailing of carrots of uniform size from respective cultivars was done with knife. Samples were washed with tap water. 50.0 grams of carrot sample was mashed and blended with 100mL of deionized water using blender. Filtration of blended sample was done through filter paper (Whatman No 1) to separate the filtrate for further analysis ((Mahmood, 2011). Demineralization of filtrate was done by passing through cat-ion and an-ion resin. Samples was filtered by using syringe filter (0.22 µm). to remove microbes. Triplicate samples were prepared and analysis was done within twenty four hours. b) HPLC system specification Saccharides analysis of samples from respective cultivars was performed by HPLC system (LC-20A, Shimadzu, Singapore). The system contained pump (LC-20AT, mobile

phase de-gasser (Model#G1322A), oven (CTO-20A/20AC) and RI-detector (RID-10A). all the systems were controlled by a software (Shimadzu-LC-solution. Light-system controller (CBM-20A/20A) was also attached to assist the system. c) Sugars analysis by HPLC Aminex-column-Bio-Rad (HPX-87K-300-7.8mm, Cat-No--1250142) attached with G- column by bio-Rad was used to separate the carbohydrates. Dematerialized water was used as mobile phase at 0.50mL/minute as flow rate. The temperature of refractive index detector was set at 40°C to separate the individual sugar contents. 20μL sample was injected through injection pore using syringe of 25μL capacity. Individual sugar contents were identified and quantified from the retention times, peak areas and calibration curves of standard sugar compounds. i.e. fructose, glucose, maltose, maltotriose and maltodextrin. d) Method validation parameters for sugar analysis

(I) Selectivity Selectivity of method was determined by using the previously published method (Zielinski et a., 2014). Briefly, the RI-detector (refractive-index-detector) was practiced to recognize the amalgams in diverse models of cultivars of D. carota. Analyte retention- times and resolutions were used for the estimation of results abstained from chromatographic-method and evaluated with the times of retention of set of standards: 0.800g/100mL of fructose, 1.400g/100mL of glucose, 0.350g/100mL of maltose, 0.250 g/100mL of maltotriose and 2.200g/100mL of maltodextrin.

(II) Linearity Method of linearity was determined by using the previously published method (Zielinski et al., 2014). Five injections (triplicate) of different-concentrations (obtained by making dilution with distilled water) of standards were used to established linearity for 5 no of sugars standards: 1.0--5.0g/100mL of fructose; 1.0--5.0g/100mL of glucose; 1.0-- 5.0g/100mL of maltose, 1.0--5.0g/100mL of maltotriose, and 1.0--5.0g/100mL of maltodextrin,. The curves of calibration for every standard were produced by scheming the area covered by the respective peak and concentration of standards.

(III) Precision The coefficient of variation (CV%) was used for the determination of intra-day- readability of the method by using the areas acquired by injecting the reproduces solutions of standard—compound-solutions {0.80g/100mL--fructose, 1.40g/100mL-- glucose, 0.35g/100mL--maltose, 0.25g/100mL--maltotriose and 2.20g/100mL-- maltodextrin} and filtered sol. Fron each cultivar of D. carota was mixed to this prepared mixture, correspondingly. Intermediate-precision was evaluated by using three replicates of prepared solutions over three nonconsecutive-days (Agência Nacional de Vigilância Sanitária, 2003) (IV) Accuracy Accuracies (inter-day) were determined in view of the revitalization attained for individual composites at unlike applications (0.25--5.25g/100mL of fructose; 0.50-- 5.50g/100mL of glucose; 0.60--5.60g/100mL of maltose; 0.80--5.80g/100mL of maltotriose, and 1.0--6.0g/100mL of maltodextrin) and be articulated via estimating the relationship among the evaluated compound quantity and results obtained (Agência Nacional de Vigilância Sanitária, 2003)

(V) Limit of quantification (LOQ) and Limit of detection (LOD)

The limit of quantification (LOQ) and limit of detection (LOD) were calculated by analytic curve method parameters, by using the following equation (Agência Nacional de Vigilância Sanitária, 2003)

3.3 10 퐿푂퐷 = 푆퐷 푋 퐿푂푄 = 푆퐷 푋 푆 푆

Where: S: slope of the analytical curve; SD: standard deviation of the response Part III 3.5 Spectrophotometric analysis 3.5.1 Sample preparation for β carotenes analysis Extraction of β-carotenes was done by the previously established method (Fikselova et al., 2008). Cutting in the form of slices having width 2-3mm and lenghth 1-1.5cm was

done with the help sharp knife. Carotenes were extracted by using ethanol and 2-propanol at 60°C. Primarily, 25.0g of D. carota sample was added to 100mL ethanol and 100mL of 2-propanol separately. Samples were heated with the help of water bath. Temperature of water bath was maintained at 25°C and 60°C during extraction of carotenes. Extraction process continue for three hours with shaking after every ten minutes and 10.0mL of sample was taken and mixed with 20.0 mL of petroleum-ether. Separation of phases was done with water and carotene phase in either was separated and diluted to 50mL 3.5.2 Determination of β carotenes by spectrophotometer For the determination of β-carotenes in prepared samples, spectrophotometer was used. Wavelength of spectrophotometer was set at 450nm and absorbance of samples was measured separately using petroleum-ether as blank. For the estimation of β-carotenes following equation was used.

Volume β − carotene = Absorbance × dilution × 1cm E1% × sample weight

Where: 1cm E1% =2592 (absorbency coefficient for petroleum ether)

3.5.3 Determination of total phenolic (TP) Folin Ciocalteu procedure, previously cited by Singleton et al. (1999)was used for the estimation of total phenolic acids. Briefly, carrot extract of 50mg (dried) from each cultivardried extract was added with 7.50mL distilled-water followed by Folin–Ciocalteu (0.5mL). The blend was potted at ambient warmth for 10.0min, and after that 1.50-mL of 20.0% of sodium-carbonate (wt/vo) be added. The blend be warmed at 40.0°C at stream bath for 20.0min moreover after that it shifted to frost-tub to chill. Aabsorb-ance be calculated by spectro-photo-meter at 755.0nm. Amounts of TP were calculated using calibration curve of Gallic acid (R2 = 0.9986). The results were described as Gallic acid mg per 100g. All samples were analyzed individually in triplicate and results were averaged and the computation be completed on the basis of F-weight. Same procedure was adopted in case of top whole and seeds of respective cultivars.

3.5.4 Determination of total ascorbic acid (TAA) For the determination of ascorbic acid contents in selected cultivars, indophenols-method was used as cited by Nielsen (1998) and the outcomes be illustrated as mg of acid- ascorbic/100g of F-weight (FW). 100g sample from individual cultivar be taken separately in glass beaker and removal of liquid be completed by way of food processor. Addition of 20mL of (1:1) acetic-acid-metaphosphoric-acid solution be done to that extractor juice & build the quantity up to 100mL with acetic-acid-metaphosphoric-acid solution. Sample-extract of 2.0mL was taken in three separate flat bottom measuring flasks (50mL) and 5.0mL of metaphosphoric-acid acetic-acid solution was added in each flask.The sample solution in flask was titrated against solution of indophenols-dye till the appearance of pink colour which persist for five seconds. The amount of dye used in the titration process was used in calculating ascorbic-acid contents in samples from respective cultivars 3.5.5 Biological activities a) Sample preparation for biological activities Extracts for the determination of biological activities were prepared by using previously cited method (Lim et al., 2007) with slight-adjustments. 25.0 g sample from each cultivar was mixed with 50mL of ethanol (75%) and homogenization was done by using an electric blender. Transferred it to volumetric flask (100mL) and made the volume upto the mark with ethanol (75.0%). Orbital shaker was used for further homogenization for twenty minutes at 200rpm. Filtration of homogenized sample was done by using muslin- cloth. The filtrate from respective cultivar was analyzed three times within 48 hours.

Same procedure was adopted in case of top whole and seeds of respective cultivars. ‘ b) Total antioxidant activity Oxidation of linoleic-acid and beta carotenes directed in theestimation oftotal antioxidantactivity by previously cited method (Taga et al., 1984). Briefly, Beta carotene (10mg) was solublized in chloroform (50mL). 3 mL of prepared sample was added in conical- flask having Tween-20 (400μL) and linoleic-acid (40 μL). Using rotary evaporator, chloroform was removed at 35ºC. 100mL of distilled water was mixed in beta carotene suspension and shacked well. Accurately, 0.2mL of prepared sample was mixed with beta carotene (3mL) solution in test tube, followed by vigorously shaking. Incubation of prepared samples in test tubes was done at 50ºC in an incubator. Spectrophotometer was tuned at 470nm for recording the absorbance values to monitor the oxidation of beta-

carotenes. Absorbance of prepared samples from respective cultivars was noted at ten, twenty, thirty, forty, fifty and sixty minutes. 0.2mL of distilled water (instead of sample) was used for the preparation of control-sample. The degradation rate of the prepared samples or extract was measured by first order kinetics.

a Sample degradation rate = ln ( ) × 1/t b where: a = absorbance at 0 min, b = absorbance at described time intervals, t = time and In=log(natural). Percent of Inhibition comparative to control was used to estimate the level of antioxidantactivity by means of following formula. The findings were reported as mg/100g of FW after calculations. Same procedure was adopted in case of top whole and seeds of respective cultivars.

Degradation of control − Degradation of sample AA% = × 100 Degradation of control c) Superoxide radical scavenging activity Superoxide radical scavenging activity of cultivars of D. carota was determined using the method described by Liu et al. (1997) and with slight modifications, briefly, 1.0mL of nitro-blue-tetrazolium (NBT) solution (phosphate buffer pH~7.4, 156μM NBT/100mM) was mixed with 1.0 mL of 468 μM NADH solution in 100mM phosphate buffer, pH 7.4, then 1.0mL of sample solution of carrot extract (30-50μg/mL) in ethanol was added and mixed thoroughly. 100μl of phenazine-methosulphate (PMS) solution was added to the above mixture to start the reaction. After that reaction mixture was incubated at 25°C for five minutes and the absorbance was measured at 560nm against blank sample. Decreased in absorbance indicated increased superoxide anion scavenging activity. d) Hydroxyl radical scavenging capacity Hydroxyl-radical scavenging-capacity (HORAC) assay was performed by using chelating-activity of metals (Fenton-like reactions) of anti-oxidants in the presence of Co

(II) complex(Ou et al., 2002). Briefly, 0.55M H2O2 solution in deionized-water was mixed with 15.70mg of CoF2·4H2O to pre[are 4.60mM Co (II) solution and 20mg of

picolinic acid (20 mg) was solublized deionized water up to 20mL (final- concentration=60.0nM,). 10.0µL of prepared sample was put/applied in the plate-reader and incubation was done at37 °C for only 10.0 min. After completing the incubation-time,

Co (II) 10.0 µL and H2O2 (10.0µL) were added accordingly,H2O2 (final- concentration=27.50mM). After gentle-shaking the prepared-samples, measurement of fluorescence was donesubsequently(after every minute). For blank-sample solution of phosphate-bufferwas used alone. For drawing the standard-curve, anti-oxidant solutions

of standard (1 ‘00, 2 ‘00, 6 ‘00, 800‘ and 1 ‘000 µM) were prepared in buffer (phosphate) having concentration (75.0mM) with adjusted at pH of 7.4. HORAC measures between standards of anti-oxidants and area (under the curve). 1µM of anti-oxidant standard consign one unit of HORAC and activity of sample was presented as mg/100g of FW. Same procedure was adopted in case of top whole and seeds of respective cultivars. e)DPPH radical scavenging assay Free-radical scavenging-activitiesof carrot extracts from each cultivar was measured by using previously cited method (Iqbal et al., 2005)withminor-modification. Fresh solution

of 2, 2 ‘-diphenyl‘ -1 ‘ -picrylhydrazyl (DPPH) was poured in1.0mL (having 25-30µg/mL dry matter) of carrot extract. Freshly prepared solution of was added to 1.0 mL of D. carota extract.Spectrophotometer (tuned at 515nm) was used for the measurement of absorbance of prepared samples from respective cultivars at 0, 0.5, 1, 2, 5 and 10min. . Unused (un- reacted amount) DPPH-radical was measured through calibration curve. Absorbance measured at 5th min was used for the comparative analysis of carrot extracts from respective cultivar. The findings of this essay were reported as DPPH scavenging capacity as mg/100g. Same procedure was adopted in case of top whole and seeds of respective cultivars. Part IV 3.6 A study for the effectiveness of carrot juice on blood parameters 3.6.1Extraction of D. carota juice D. carota (T29) of uniform size were obtained from the pasture of vegetable-section of Ayub-Agriculture Research-Institute (AARI) Faisalabad, Pakistan. Samples were topped, tailed, washed thoroughly and scrapped before extraction of juice. The extraction of juice was done by Juicer machine, Philips (model # HL7715/00, 700-Watt).

3.6.2 Subjects In this study, twenty males of variable age groups were selected from an industry in the locality of Faisalabad, Pakistan. (n = 20). Subjects were divided into four groups as per their age and 250mL of D. carota juice was given to every member of each group for a month (30 days)

3.6.3Experimental groups The experimental groups of individuals were established on the basis of age, the detail is given below. Treatment group No 1 This group members (n= 5) were having age (20 to 30 years) Treatment group No 2 This group members (n= 5) were having age (31 to 40 years) Treatment group No 3 This group members (n= 5) were having age (41 to 50 years) Treatment group No 4 This group members (n= 5) were having age (51 to 60 years)

Each subject (group wise) was asked to drink fresh D. carota juice daily for thirty days. Subjects signed a consent form to agree the rules and regulation of the study and were agreed to drink carrot juice as per study rules. D. carota juice was delivered to research participants daily in the morning. BMI status was studied by measuring height and weight of the participants both pre-test and post-test. 3.6.4 Collection of blood samples A licensed male nurse was agreed to collect the fasting blood samples at the start of the study and after thirty days. The test tubes were used for the collection of blood samples and centrifuged at 1500 rpm for fifteen minutes. 3.6.5 Blood analysis The blood chemistry i.e. sodium, potassium, calcium , albumin, Glucose, blood cells, hemoglobin, hematocrit, platelets and lipid profile were measured by using Microlab 300 Lx/Chemistry Analyzer, France and Hematology analyzer, EKSV (model noEKSV-2300, China) from Prime Care Clinical Computerized Lab., Faisalabad, Pakistan,. Blood

pressures were recorded by using apparatus OMRON Model no HEM 711 at the beginning and end of this research. The blood pressures were repeated in case of reading error or a participant seemed nervous. 3.6.6 Total antioxidant status The plasma was separated from blood by using centrifuge machine at 1500rpm and an aliquot was refrigerated and total antioxidant status was measured by using a commercially available kit (Calbiochem, San Diego, CA, USA). 3.6.7 Malondialdehyde status The plasma was separated from blood by using centrifuge machine at 1500rpm. Separated blood plasma was tested for quantitative measurement of malondialdehyde (MDA) by using lipid per oxidation (MDA) assay kit (Vancouver, WA, USA). 3.8 Statistical analysis From respective-cultivar, three samples were collected for analysis. Triplicate analysis was done of individual sample from each cultivar and numeric- findings were recorded as mean (N = 01.0 x 03.0 x 03.0) ± SD (N = 01.0 × 03.0 ×03.0). To assess calibration curves for sugar standards, linear-models were used. Analysis of variance i.e. ANOVA was used for the determination of lacks of fit and regression equation were determined (p ≥ 0.05) and level of significance (p < 0.05). ANOVA i.e. analysis of variance, regression coefficient and Probability values were determined by using software (statistical). 2000- Mini-tab 13.2-Version (Mini-tab-Inc--Pennsyl-vania-, U.S.A).

Chapter 4 RESULTS AND DISCUSSION In Pakistan, different sorts of root vegetables are accessible regularly in specific seasons, however are not consumed to the degree these ought to be, inspite of their high nutritive esteem. Investigation to explore theimperativeattributes of new cultivars of D. carota can be utilized to surmount the nutritional-disorders. Part I Proximate composition is a type of scientific analysis made to find out the estimated quantities of certain substances within the mother material (Coultate, 2009). This practice is being exercised by many scientists to study such types of substances in animal feed, bio-fuels, coal, fruits, vegetables etc. It is a complicated process, often involves many types of extraction protocols for different types of materials. These informations are used, in quality controlling of various products, for ensuring hazardous materials and to determine the healthy ingredients for living beings (Duley, 2012). 4.1Proximate composition 4.1.1 Proximate composition of seeds The data regarding proximate composition of Daucus carota seeds is presented in Table 4.1. DCY cultivar showed higher moisture contents (7.8%) and that of lowest (6.1%) in DCW cultivar. Statistical-analysis proved that moisture (%) varied significantly (ρ < 0.05) among cultivars. The findings of the present study regarding the moisture contents are comparable to the findings of Musa and Chalchat (2007) for carrot seeds native to local market of Turkey. The earlier scientific studies has approved that low moisture contents are good for long-term storage of seeds (Ellis & Hong, 2006). The protein contents in seeds of selected cultivars of D. carota varied from 21.5% for DCW to 24.6% for T29. The higher protein contents in seeds of D. carota proved the fact that higher protein contents in vegetable seed have not only major role in genetic delivery system and biodiversity of plants but also a good food source for humans and animals (Job & Bailly, 2013). Statistical analysis regarding protein contents in seeds of selected cultivars varied significantly (ρ < 0.05). The data regarding oil contents in seeds of selected D. carota cultivars is presented in Table 4.1. The oil contents in seeds of selected cultivars D. carota varied from 5.9% for DCY to 7.8% for T29 cultivar. Statistical analysis revealed that oil

contents in seeds of D. carota assorted considerably (ρ < 0.05) along with cultivars. The finding regarding oil contents extracted from T29, DCR, and DC3 are comparable to the findings (7.89%) of previously published work (Musa & Chalchat 2007) but DC90, DCY, DCP and DCW have comparatively lower oil contents. The difference in results regarding oil contents extracted from carrot seeds in both studies may be due to difference in species, extraction methods, operating parameters and moisture contents (Orhevba et al., 2013; Ademola et al., 2016). The data regarding the carbohydrates contents in seeds of selected cultivars of D. carota is presented in Table 4.1. The carbohydrate contents in seeds of D. carota cultivar varied from 46.8% for DC3 to 51.4% for DCW cultivar. Statistical analysis revealed that carbohydrates contents in seeds of D. carota diverse considerably (ρ < 0.05) between cultivars. The carbohydrates contents in selected cultivars are lower than other vegetable seeds (Ferguson et al., 1991) like barley (76%), oat (66%) and wheat (67%) but higher than soybean (17%). The difference in carbohydrates contents among the cultivars of present study is due to difference in cultivars and among both studies is due to the nature of seeds under investigation. The considerable carbohydrates contents in selected cultivars of D. carota proved the fact that these are good source of energy (German & Dillard, 2006). The selected cultivars of carrot in the present study are also a good source of crude fiber which varied from 1.8 % for DCR to 4.1% for DCW presented in Table 4.1. Statistical analysis regarding the crude fiber contents diverse considerably (ρ < 0.05) amongst cultivars. The results related to crude fiber contents in seeds of selected cultivars of D. carota (T29, DCR, DC90) are comparable to crude fiber contents of (1.87-2.13%) in seeds of five types of carica papaya (Nwofia et al., 2012) on the other hands DC3, DCY, DCP and DCW showed higher fiber contents. The difference in results in both studies is due to the difference in nature of seeds under study. The crude fiber contents proved the fact that carrot seeds have much potential for the remedial action for stomach disorders (Adejumo et al., 2005). The data regarding the ash contents in seeds of selected D. carota cultivars presented in Table 4.1. The ash contents varied from 8.9 % for DCW to 11.3 for DCY. Statistical analysis regarding ash values diverse considerably (ρ < 0.05) amongst cultivars. The higher ash contents in seeds of selected cultivars proved the fact that high ash contents in seeds give them medicinal properties (Hannah & Krishnakumari, 2015). In view of the above proximate composition, it may be concluded that seeds of selected D. carota cultivars have both the food and medicinal properties, which may be used for

the production of value added products and functional foods by process and manufacturing industries. 4.1.2 Proximate composition of carrot roots Exploration of valuable nutrients of novel cultivars of carrot can attract the attention of diet conscious consumers. The data regarding the proximate composition of the selected cultivars of fresh D. carota is presented in Table 4.2. DC90 cultivar showed higher moisture contents (91.0%) and that of lowest (86.6%) by DCP among the selected cultivars. A significant difference (ρ < 0.05) in moisture contents was observed among novel cultivars of D. carota. The findings of present study are higher to the moisture contents of red carrots (84.23%) presented by Shyamala and Jamuna (2010). The earlier scientific studies has approved that the activation of enzymes and co-enzymes to perform metabolic functions is also associated with high moisture (Iheanacho & Udebuani, 2009). It could be concluded from the findings related to high moisture contents that new cultivars of carrot may be a good diet for improving the enzymatic and metabolic processes in living beings. It is scientifically approved that proteins in D. carota cultivars are recognized as anti-freezing protein which have ability to protect carrotsfrom drawstring conditions of environment and weather(Smallwood et al., 1999). Our finding regarding the proximate composition of different cultivars of D. carota proved the cultivar DCP with highest protein contents (1.6%) and DC3 with that of lowest (0.59%). Statistical analysis revealed that total protein contents in selected cultivars diverse considerably (ρ < 0.05) between the cultivars. Finding of present study regarding the protein contents in T29, DCR, DC3, DC90, DCY, and DCW, cultivars are not comparable to that of (1.0g/64g or 1.56 %) for previously investigated cultivars of carrots (Hongu et al., 2015) except DCP cultivar having protein 1.60%. The findings related to protein in selected cultivars of D. carota prove the fact that vegetables are poor source of proteins (Skov et al., 1999) and can be used to reduce the severity of kidney degeneration in human beings (Fouque & Laville, 2009). It was observed that the highest fat contents (0.42%) were found in DCW and that of lowest (0.3%) in T29. Statistical analysis showed significant (ρ < 0.05) differences in fat contents among cultivars. The fat contents of all the new lines are to much lower than the previously reported findings (2.43% fat) in carrot powder (Gazalli et al., 2013). The difference in fat contents might be due the genetic makeup new cultivars and extraction

procedure (Bourn & Prescott, 2002). The low fat contents in D. carota confirmed that vegetables are poor source of fats (Ejoh et al., 1996). Kris-Etherton et al. (2002) reported that 1.0 - 2.0% fat can fulfill the body requirements for healthy living sufficiently. They, further, claimed that excess fats in foods may cause cardiovascular issues, so it is important to develop new vegetables and fruits with low fat contents. The data regarding carbohydrate contents in the investigated cultivars showed that DCR cultivar was found to have high carbohydrate contents (8.0%) and that of lowest (6.6%) in T29. Statistical analysis revealed that the carbohydrate contents diverse considerably (ρ < 0.05) between the cultivars. The findings of present study regarding the carbohydrates are on lower side than that of (9.38%) for previously studied cultivar of carrot (Hongu et al., 2015). The low carbohydrate contents in newly developed cultivars proved that these have low calorific energy values. The calorific values of new cultivars of D. carota were comparable to vegetables with low calorific energy values (Lintas, 1992). The carbohydrate contents in newly developed cultivars are much lower than the recommended dietary allowance (RDA) contents for carbohydrates (210-130g/100g) for normal human being (Patricia et al., 2014). It could be concluded that the carrots contained insufficient amount of carbohydrates which is not enough for a normal-human requirements for healthy living. However, it is excellent food for diet conscious (weight conscious) community. It is quite reasonable to recommend it as remedy for the people suffering from obesity. The data regarding crude fiber contents in the newly developed cultivars showed that DCW cultivar was found to have high crude fiber contents (3.2%) and that of lowest (1.6%) in DC90. Statistical analysis revealed that the crude fiber values diverse considerably (ρ < 0.05) between the cultivars. The findings of present study regarding crude fiber in case of DCR, DC90, DCY, DCP and DCW are not comparable to the fiber contents (2.39%) in previously investigated raw carrots (Li et al., 2002) but are quite comparable to T29 (2.2%) and DC3 (2.3%) . The difference in findings regarding crude fiber in the present study is due to the difference in cultivars and among both studies it may be due to the difference in cultivars or determination method. The foods having high fiber contents are useful to remedies and regulate the stomach problems (Slavin et al., 2013). It could be concluded that DCW cultivar having high crude fiber could be recommended for healthy digestion process.

Table 4.1 Proximate composition of Daucus carota cultivars seeds

Contents Cultivars of Daucus carota (% db) T29 DCR DC3 DC90 DCY DCP DCW

Moisture 7.3±0.82e 6.9±0.91d 7.1±0.59c 7.3±0.87f 6.1±0.88a 6.4±0.71b 7.8±0.69g

Protein 24.6±1.1g 23.6±1.3d 22.9±1.4b 24.5±1.1f 23.8±1.2e 22.9±1.7c 21.5±1.3a

Oil 7.8±0.56g 7.9±0.52f 7.6±0.60e 6.9±0.40d 5.9±0.41a 6.1±0.31b 6.4±0.55c

Carbohydrates 47.8±3.1b 48.9±2.8e 46.8±3.0a 49.2±2.2d 48.6±2.6c 50.2±3.1f 51.4±2.9g

Crude fiber 2.1±0.12b 1.8±0.14a 3.1±0.15c 2.2±0.12d 3.7±0.12f 3.5±0.12e 4.1±0.14g

Ash 9.9±0.43b 10.2±0.49d 11.2±0.53e 10.2±0.48c 11.3±0.56g 11.2±0.59f 8.9±0.41a

The values are expressed in a-gmean ± standard deviation followed by the same letter, within a row, are not significantly different (ρ > 0.05). d.b means values are calculated on dry basis.

Ash contents of newly developed cultivars are presented in Table 4.2. The DCP cultivar was found to have high ash contents (1.12%) and that of lowest (0.42%) in T29. Statistical analysis revealed that ash values diverse considerably (ρ < 0.05) amongst the cultivars. The findings regarding ash contents in newly developed cultivars are on lower side in case of T29, DCR, DC3, DC90 and DCW as comparable to previously published results but ash contents in DCP (1.12%) and DCY (1.11%) cultivars are quite comparable to ash contents (1.027%) in red carrots (MotegaonkarManorama & SalunkeShridar, 2012). The variation in ash contents in both studies may be due to the variation in species, agro-climatic conditions, age of plant and cultivation stages (Gupta et al., 2005). DCY and DCP containing higher ash contents among newly developed cultivars could serve as mineral supplementation. The ash consists of minerals which play a significant role in controlling major physiology of body (Anjorin et al., 2010).

4.1.3 Mineral profiles of selected cultivars of carrot The minerals play a significant role in controlling major body functions and deficiency of one of these minerals in certain body organs results in physiological irregularities in biological processes (Anjorin et al., 2010). As a part of nutritional probing, it’s very important to explore the mineral composition of newly developed carrot cultivars. Mineral profile of D. carota is presented in Table 4.3. It was observed that highest cobalt contents (0.45µg/g) were found in DCP and that of lowest (0.21 µg/g) in DC90 cultivar. Statistical analysis revealed that cobalt contents in selected cultivars diverse considerably (ρ < 0.05) between the cultivars. The findings of present study regarding cobalt contents in T29, DC90, DCY, DCP and DCW cultivars are not comparable to that of (0.32 µg/g) for previously studied cultivar of red carrots (Al-Farhan, 2013), in contrast to DCR (0.32 µg/g) and DC3(0.34 µg/g) cultivars, having comparable results. The variation in results in both studies is due to difference in cultivars. Cobalt is a fundamental component of vitamin-B12 which is imperative for cell operations in living beings (Trowbridge & Martorell, 2002). It may be concluded from the findings related to cobalt contents that selected cultivars can be used in value added products to remedies the deficiency of vitamin B12. Zinc (Zn) regulates the function of immune-system, digestive-system, reduces stress, controls diabetes, reduces hair loss and helpful during pregnancy (Bhowmik & Chiranjib, 2010). The highest Zn contents (3.12µg/g) were found in DCP and that of lowest (2.04 µg/g) in DCW cultivar. Statistical analysis revealed that Zn contents in

selected cultivars diverse considerably (ρ < 0.05) between these varieties. The findings of present study regarding Zn contents are higher to that of (2.21µg/g) for previously developed cultivar of red carrots (Trowbridge & Martorell 2002) but DCW cultivar showed comparable (2.04 µg/g) results. They also reported that zinc (Zn)plays an active role in the synthesis of chlorophyll andstimulates enzymes. It also plays an important role in the synthesis of starch, chloroplast and auxin. Copper (Cu) is an essential element for the growth of human body, increase the utilization of iron, regulate enzymatic reactions, and decrease premature aging (Qureshi et al., 2005). It was observed that highest copper (Cu) contents (1.96µg/g) were found in DCP and that of lowest (1.23 µg/g) in DC90 cultivar. Statistical analysis revealed that Cu contents in selected cultivars diverse considerably (ρ < 0.05) between cultivars. Copper (Cu) is an essential constituent of several redox and biosynthetic-lignin enzymes (Williams, 1992). Fe contents in newly developed cultivars are presented in Table 4.3. It showed that highest Fe contents (5.79µg/g) were found in DCP and that of lowest (4.31 µg/g) in DC90 cultivar. Statistical analysis revealed that Fe contents in selected cultivars diverse considerably (ρ < 0.05) between cultivars. The findings of present study regarding Cu and Fe contents in some of the cultivars studied in present study are not comparable to that of (1.51µg/g) and (4.68µg/g) respectively for previously developed cultivar of red carrots (Al-Farhan , 2013). The difference in FE and CU contents is may be due to difference in cultivars and detection methods, soil and water quality (Jamali et al., 2009). Function of strontium (Sr) ion is not defined in human body but strontium ranelate is used in the treatment of postmenopausal osteoporosis (Marie, 2005). The data regarding strontium contents in newly developed cultivars showed that highest strontium contents (4.13µg/g) were found in DCP and that of lowest (3.0 µg/g) in DCR cultivar. Statistical analysis revealed that Strontium contents in selected cultivars diverse considerably (ρ < 0.05) with cultivars. The findings of present study regarding Sr contents are on lower side to that of 4.68µg/g, for previously developed cultivar of red carrots (Al- Farhan, 2013). According to “recommended dietary allowance” (RDA) specifications for iron (15.0 mg per day), copper (2.0mg per day), cobalt (1.0mg per day), zinc (15.0 mg per day) and Strontium (not defined) and for healthy young human (Cooperman et al., 2003). In view of RDA recommendations most of the cultivars investigated in the present study could be an excellent source of minerals which are very useful for maintaining and curing health issues. It may be concluded that selected cultivars of D. carota having

remarkable contents of essential minerals can be used in the preparation of functional food, not only on consumer level but also on commercial scales in process industries.

4.1.4 Proximate composition of top whole of D. carota cultivars During development stages of vegetable leaves, chlorophyll plays a significant role in the synthesis of nutrients like protein, carbohydrates and micronutrients (Rajcan et al., 1999). The top wholes of selected cultivars of D. carota were investigated for proximate composition to explore its vegetal and food attributes. The data regarding the proximate composition is presented in Table 4.4. Moisture contents varied from 88.67% for DC3 to 92.98 for DCR cultivar. A significant difference (ρ < 0.05) in moisture contents was observed among top whole of D. carota cultivars. Findings of present study related to moisture contents in carrot top whole are not comparable to previously published results (Patricia, et al., 2014) for other commonly consumed vegetables like Andasonia digitata (77.63%), Amaranthus hybridus (72.98%) and Ceiba patendra (70.45%), but results regarding the moisture contents in both studies are in agreement with recommended values in leafy vegetables (60 t0 90%) reported by FAO (2006). The variation of the results is due to the difference in types of vegetable. It has been investigated that high moisture contents in leafy vegetables are beneficial for survival in drastic and aired conditions of weather (Agwu, 2015). It may be concluded that top wholes of selected cultivars of D. carota has vegetal properties like other leafy vegetables and has ability to overcome the drastic conditions of the weather. The protein contents in top whole of selected cultivars varied from 18.5 for DC90 to 21.2 % for DCY cultivar. Statistical data related to protein values speckled considerably (ρ < 0.05) with cultivars. The protein values in top whole of selected cultivars are not comparable to other leafy vegetables like bitter leaf (50.64%), India spinach (58.80%) and scent leaf (62.71) reported by Asaolu et al. (2012). Findings related to protein contents in present study proved that novel cultivars are potent source of proteins as per body requirements. About 18.60 to 30.92% proteins are recommended for pregnant and lactating mothers (FND, 2005) and carrot top whole of D. carota have much potential to overcome the needs of protein for body requirements.

Table 4.2 Proximate composition Daucus carota cultivars roots

Contents Carrot cultivars

%age T29 DCR DC3 DC90 DCY DCP DCW

Moisture 90.6 ± 1.81c 88.8 ± 1.79d 90.6 ± 2.10b 91.0 ± 1.89a 87.9 ± 1.88f 86.6 ± 1.93g 87.9 ± 2.00e

Protein (d.b) 1.1 ± 0.04d 1.0 ± 0.06f 0.59 ± 0.03g 1.1 ± 0.02e 1.2 ± 0.03c 1.6 ± 0.08a 1.2 ± 0.05b

Fat (d.b) 0.30 ± 0.03g 0.36± 0.02f 0.39 ± 0.04d 0.41 ± 0.03b 0.40 ± 0.02c 0.36 ± 0.03e 0.42 ± 0.04a

Carbohydrates (d.b) 6.6 ± 0.16g 8.0 ± 0.13b 6.8 ± 0.15f 7.1 ± 0.13d 7.2 ± 0.14c 7.9 ± 0.16a 6.9 ± 0.18e

Crude fiber (d.b) 2.2 ± 0.06e 2.0 ± 0.04f 2.3 ± 0.06d 1.6 ± 0.05g 2.6 ± 0.07c 3.1 ± 0.08b 3.2 ± 0.08a

Ash (d.b) 0.42 ± 0.02g 0.80 ± 0.03d 0.72 ± 0.04e 0.81 ± 0.05c 1.11 ± 0.08b 1.12 ± 0.08a 0.69 ± 0.05f

Calorific energy 28.76 ± 2.1g 34.01 ± 3.5b 28.98 ± 2.6f 31.46 ± 2.3e 31.98 ± 2.9c 35.12 ± 3.3a 31.08 ± 2.8d (kcal/100g) The values are expressed in a-gmean ± standard deviation followed by the same letter, within a row, are not significantly different (ρ > 0.05). d.b means values are calculated on dry basis

Table 4.3 Mineral composition of D. carota roots

D. carota cultivars Minerals (µg/g) T29 DCR DC3 DC90 DCY DCP DCW

Cobalt (Co) f0.27±0.01 e0.32±0.02 d0.34±0.03 g0.21±0.02 c0.36±0.01 a0.45±0.03 b0.37±0.02

Copper (Cu) e1.39±0.02 d1.41±0.02 f1.33±0.02 g1.23±0.03 b1.46±0.02 a1.96±0.03 c1.43±0.04

Iron (Fe) e4.41±0.04 d4.49±0.03 g4.05±0.04 f4.31±0.05 b4.93±0.07 a5.79±0.11 c4.86±0.08

Strontium (St) d3.41±0.03 g3.00±0.04 e3.38±0.03 f3.12±0.04 b3.66±0.03 a4.13±0.04 c3.46±0.06

Zinc (Zn) f2.41±0.02 b2.87±0.03 c2.69±0.02 e2.43±0.03 d2.65±0.02 a3.12±0.03 g2.04±0.04

The values are expressed in mean ± standard deviation. The superscript alphabets in the row show significant difference (ρ < 0.05).

Top whole of selected cultivars of D carota showed highest oil contents (1.92%) \in DCR and that of lowest (2.56%) in DCP cultivar. Statistical data related to oil values diverse notably (ρ < 0.05) between cultivars. The findings related to oil content are not comparable to other leafy vegetables (4.9%) but oil contents in carrot top whole falls within range ((1.17 to 4.90%) reported for other commonly eaten vegetables, which proved the fact that vegetables are poor source of oil or lipids (Ejoh et al., 2007). It can be concluded from these findings that carrot top whole have oil contents like other leafy vegetables can be used for cooking and eating purposes alone or in daily servings. Carbohydrates in fruits and vegetables are the source of energy (Joshipura et al., 2009). The carbohydrates contents in selected cultivars of present study varied from 2.91% for DC90 to 3.61% for DCP cultivar. Statistical data regarding the carbohydrates findings diverse notably (ρ < 0.05) amongst cultivars. Findings related to carbohydrates in top whole of D. carota cultivars are not comparable to the findings (1.1g/30g) in spinach (Stamm- Griffin et al., 2003). The difference in result in both studies is may be dot to difference in reporting units or difference in type of vegetable under study. The presence of lower carbohydrate contents like other vegetables revealed the fact that carrot top whole can be used as vegetable like other leafy vegetables. The finding regarding to crude fiber contents varied from 1.93% for DCR to 2.49% for DCP cultivar. Statistical analysis regarding crude fiber values diverse notably (ρ < 0.05) with cultivars. The crude fiber contents in top whole of cultivars under study are not comparable to previously cited results (1.86-14.71%) in different vegetables native to Pakistan (Hussain et al., 2009), but Allium sativum having comparable results (1.86%). The difference in result is due to the difference in types of vegetable under investigation. The findings of present study related to crude fiber contents are comparable to previously published conclusion for leafy vegetables that carrot top whole is poor source of crude fiber (Venkataramanan et al., 2015). The top whole of selected cultivars of D. carota showed that highest ash contents (8.71%) were found in DCP and that of lowest (11.45%) in DC90 cultivar. Statistical data regarding ash values diverse considerably (ρ < 0.05) amongst verities. The findings related to ash contents are not comparable to ash contents of many of leafy vegetables like Spinacia oleraceae (23.97%), Allium sativum (4.84%), Amaranthus viridus (22.84%) and

Chenopodium album (22.15%) native to Pakistan (Hussain et al., 2009), in another study Patricia et al. (2014) reported comparable in different vegetables (7.25 to 26.79) reported in leafy vegetables (Patricia et al., 2014). Higher ash contents have proved the fact that carrot top whole have significant potential of minerals (Rupérez, 2002). From the findings related to

proximate composition, it may be concluded that carrot top whole of all the cultivars investigated in the present study have much potential not only as a vegetable as human nutrition but also as a fodder for animal feeding. 4.1.5 Physcico-chemical properties of carrot seed oil Carrot seed has cytotoxic properties against gastric lines (Ahmed, et al., 2005). It is carminative, diuretic, cytophylactic, antiseptic, emmenagogue, vermifuge and depurative in nature (seller, 2005). The data regarding the physic-chemical properties are presented in Table 4.5. Acidy measurement is a quality parameter for edible oil, oil having low acidy are considered to be better in quality. Acidy of seed oil depends upon the humidity of the environment, higher the humidity higher will be the acidity of oil (Gunstone, 2011). The data regarding acidity (oleic acid) of carrot seed oil of selected D. carota cultivars is presented in Table 4.5. Acidity of carrot seed oil ranged from 4.9% for DC3 to 5.6% for DCP, DCW and T29 cultivars. Statistical analysis regarding acidity diverse considerably (ρ < 0.05) with cultivars. The findings related to acidity is less than recommended acidity (<10%) for crude edible oil (Ozcan et al., 2007). From the findings regarding acidity of seed oil of selected cultivars of D. carota, it may be concluded that the seed of these cultivars can be used for commercial production of edible oil. Determination of peroxide value is a quality parameter of oil and evidence about rancidity (primary oxidation) in oils and fats (Boskou, 2015). The data regarding the peroxide value is presented in Table 4.5. Peroxide value in selected cultivars of D. carota varied from15.0 meq/Kg for DCP to16.0 meq/Kg for DCY. Statistical analysis showed significant (ρ < 0.05) differences in peroxide values among cultivars. The findings regarding the peroxide contents in selected cultivars of D. carota are in agreement with peroxide value requirements for commercial edible oils which is >10meq/g (Miller, 2012). The data regarding the peroxide values revealed that seeds of selected cultivars of D. carota investigated in the present study may be used for extraction of oil for edible purposes. Saponification number of oils and fats gives information about the characteristics of fatty acids by defining the carbon length and molecular weight of certain fatty acids. The long chain fatty acids in oil and fats showed low saponification values (Co et al., 2011). The data regarding the saponification number is presented in Table 4.5. Saponification values in seed oil of selected cultivars of D. carota ranged from 141.4 for DCR to 149.7 for DCP cultivar. Findings regarding the saponification number of seed oil of D. carota cultivars are comparable to saponification values (90-140) of sweet almond oil and hydrogenated

vegetable oil (Saponification chart, 2017). Statistical analysis regarding the saponification number showed significant (ρ < 0.05) differences among seed oil of D. carota cultivars. Findings regarding the saponification number showed that carrot seed oil have similar properties like almond and vegetable oils, which may be use for edible purposes like other edible oils. Unsaponifiable matter is a quality parameter during the selection of oil for soap formation. It is beneficial in soap formation, having properties like moisturization, conditioning and texture. Large quantities of unsaponifiable matter my results in inferior quality soaps (Gunstone et al., 2007). The data regarding unsaponifiable matter presented in Table 4.5. The unsaponifiable matter in seed oil of selected D. carota cultivars ranged from 8.92g/Kg for DCR to 10.2g/Kg for DCP cultivar. Statistical analysis regarding unsaponifiable matter diverse notably (ρ < 0.05) with cultivars. The findings related to unsaponifiable matter of the present study are comparable to unsaponifiable matter (6-17%) of unrefined shea butter (Gunstone et al., 2007), used in soap formation. From the findings of the present study it may be concluded that seed oils of selected D. carota cultivars have significant potential for manufacturing of moisturizing and conditioning soaps on commercial scales. Specific gravity is a quality characteristic of oil and is a simple mean to obtain the concentration of various substances (Hough et al., 2012). The data regarding the specific gravity of seed oils of selected D. carota cultivars is presented in Table 4.5. Specific gravity of seed oils varied form 0.972 for T29 to 0.991 for DCR cultivar. Statistical analysis revealed the significant (ρ < 0.05) differences in specific gravity among cultivars. The findings regarding the specific gravity of seed oil of D. carota cultivars are slightly different from previously reported results (0.9811) in carrot seed oil collected from local market of Turkey (Ozcan et al., 2007). The little difference in specific gravities is due to the difference in cultivar or environmental conditions under which measurements took place. Form the findings of present study regarding specific gravity it may be concluded carrot seed oil could be used for comparative standardization of other liquids. Refractive index is also an analytical tool for the determination of oil quality; it is used to determine the degree of unsaturation of oil (Andrikopoulos et al., 2002). The data regarding the refractive index of seed oil of selected D. carota cultivars is presented in Table 4.5. Refractive index of extracted seed oils varied from 1.4562 for DCW to 1.5321 for DCP cultivars. Statistical analysis revealed the significant (ρ < 0.05) differences in refractive index among cultivars. The findings regarding to refractive index of the present study are comapreable to the previously published result (1.473) of carrot seed oil collected from local

market of Turkey (Ozcan et al., 2007). On the other hand refractive index of carrot seed oils of present study is also comparable (~1.4700) to other edible oil (Firestone, 1999). From the findings of present research regarding refractive index it may be concluded that carrot seed oil can be used, not only for edible purposes after refining but also blends with other oil, may be beneficial for the oil processing industries. Data regarding physico- chemical characterizes of seed oil of selected D. carota cultivars showed that carrot oil has comparable physic-chemical properties to commercial edible oils. It may be concluded that carrot seed oil of all the selected cultivars can be used not only for edible purposes after refining but also for the formation of conditioning soaps on commercial scales. Many scientists has already recommended that blending of oil can enhance the oxidative stability under long-term storage (Chatha et al., 2011), proving the fact that carrot seed oil, having antioxidant properties may be used for blending with vegetable oil to enhance its functionality and oxidative stability.

Table 4.4 Proximate composition of Daucus carota cultivars top whole

Cultivars of Daucus carota Contents %

T29 DCR DC3 DC90 DCY DCP DCW

Moisture 91.34±2.1e 92.98±3.2g 88.67±2.2a 91.5±3.1f 90.2±2.1c 91.18±2.2d 89.2±2.0b

Protein (db) 19.5±1.3c 21.2±1.5e 20.8±1.7d 18.5±1.4a 21,2±1.8f 18.9±1.6b 19.3±1.5c

Oil (db) 2.32±0.16b 1.92±0.1.9a 2.55±0.21f 2.33±0.18d 2.37±0.13c 2.56±0.17g 2.45±0.18e

Carbohydrates (db) 3.31±0.21f 3.11±0.19e 2.98±0.22b 2.91±0.27a 3.02±0.23d 3.61±0.28g 3.11±0.10c

Crude fiber (db) 2.02±0.10c 1.93±0.14a 2.20±0.14f 1.98±0.12b 2.18±0.13e 2.49±0.16g 2.1±0.15d

Ash (db) 10.67±1.1d 11.34±1.05f 10.93±0.93e 11.45±1.12g 9.7±1.05c 8.71±1.21a 9.3±1.11b

Table 4.5 Physico-chemical properties of Daucus carota cultivars seed oil

Cultivars of Daucus carota Parameters

T29 DCR DC3 DC90 DCY DCP DCW

Acidity (oleic acid 5.6±1.12e 5.1±1.3c 4.9±1.31a 5.1±1.2b 5.3±1.33d 5.4±1.32e 5.3±1.42e %)

Peroxide value 15.5±1.81c 16.1±1.66f 15.8±1.81e 14.9±1.67d 16,0±1.78g 15.0±1.43a 15.2±1.76b (meq/Kg)

Saponification 144.8±12.4c 141.4±11.9a 147.6±13.1e 145.8±13.5d 148.9±14.1f 149.7±14.6g 141.9±11.9b number

unsaponification 9.7±1.42f 8.9±1.11a 9.4±1.53c 9.5±1.56d 10.2±1.61g 9.8±1.29e 9.1±1.48b matter (g/kg)

Specific gravity 0.972±0.001a 0.991±0.002g 0.976±0.001b 0.981±0.002e 0.981±0.001d 0.990±0.002f 0.978±0.001c

1.4681± 1.4732± 1.4891± 1.4591±. 1.4902± 1.5321± 1.4562± Refractive Index 0.0002c 0.0002d 0.0003e 0003b 0.0004f 0.0006g 0.0003a

Part II 4.2 Quantification of individual compounds 4.2.1 Quantification of individual sugar compounds Linear-regression parameters obtained from the sugar standard calibration curves are given in 2 2 Table 4.3. The findings of R i.e. regression--coefficient and R adj i.e. adjusted regression coefficient near to one show that model elucidate every unevenness of retort-data approximately its mean. Probability values obtained for consequence of sculpt & normality point out the altitude of consequence (Table 4.3), which are analogous to the findings (R2 = 0.999, p < 0.0001) as illustrated by Lakka & Reddamoni. (2014). Range & linearity of appliance of procedure were designed by sketching the cali-bration-curve by linearmodels of glucose, fructose, maltose, maltodextrin & maltotriose correspondingly. The variation-coefficients (CV %) of the calibration-standards (n = 5)ranged from 0.24 - 0.98%,forrepeatability(intra-day) andintermediate-precision(inter-day) 0.31and 2.55%, respectively (Table 4.4). According to the Brazilian legislation for validation of bio-analytical &analytical techniques, the CV% should be less than five percent (Zielinski et al., 2014). The calculated values of limit of quantification (LOQ) and limit of detection (LOD) for standards evaluated which varied from 49.36 to 178.23 mg/L and 48.13 to 59.45mg/L correspondingly (Table 4.4). LOQ and LOD are analogous to Muir et al. (2009) for the sugar column of 250 mg/L and 1.00 to 50.00 mg/L respectively. The recovery levels of saccharides like maltotriose, fructose, maltose, glucose and maltodextrin were estimated in test-sample having different contents of the standards added to all samples of D. carota cultivars (Table 4.5). The determination of recovery level of analytical method is a tool for the confirmation of method validation and accuracy (Feinberg, 2007). DCP cultivar showed the highest recovery of fructose with lowest concentration of fructose standard. The DC90 showed the lower recovery of maltodextrin with highest concentration of maltodextrin standard. In case of glucose, DCY showed the higher recovery with low as well as with high concentration of glucose standard. But overall trend in case of all the standards, recovery rate is higher with higher concentrations of respective standards. These results are comparable to Ribani et al. (2004) which described that the dispersal of calculated-results increases with the decrease in the standard-concentrations added in samples from respective cultivars.The findings regarding method validation of the present research are in acceptable (satisfactory) limits recognized by Brazilian-legislation for the substantiation of chromatographic-methods (Zielinski et al., 2014).

The equally formulated technique was used to conclude the saccharides in test- samples of the 7 D. carota verities. Figures 4.1- 4.8 showed the typical chromatograms of sugars present in all the cultivars and the intensity of sugars in de-ionized test-samples are shown in Table 4.6. The DCP cultivar showed higher total sugar contents (6.311 g/100g) and DC90 showed comparatively lower level of overall sugar conc. (4.488 g/100g). Beside major sugars, some minor sugars (maltotriose and maltodextrin) are also determined in all cultivars of D. carota, which are not reported in literature. Alasalvar et al. (2001) and Alabran et al. (1973) dogged the sweetie substances in diverse verities of D. carota, they determined that foremost sugars there in D. carota are Sucrose and fructose. The contents of maltotriose, fructose, maltose, glucose, and malto-dextrin in different cultivars of D. carota are shown in Table 4.6. Mal-tose was the major sugar in each of 7 verities of D. carota (01.886--02.463g/100g), tag on by fructose (01.103--02.09g/100g), glucose (0.9630--1.3950g/100g). The concentration of maltodextrin and maltotriose were ranged from 0.1660--0.5830g/100g and 0.0040--0.0580g/100g respectively in all cultivars of D. carota. Statistics showed a significant difference (p < 0.05) among sugar contents of different concentrations. Simon et al. (1980) estimated sweetie conc. valued from 03.0 to 08.0%, by sucrose prevailing and smaller quantities of fructose & glucose. In the present study maltotriose and maltodextrin were determined first time as novelty sugars in D. carota cultivars. Maltodextrin and malotriose have the ability to protect membranes, proteins and the electron transport chain of photosynthesis under freezing and heat stresses (Kaplan & Guy 2004; Weise et al., 2004). The sugar conc. of D. carota are prejudiced by geno-type and atmosphere. Proof for sturdy inherent-power of whole sugar values is cheering in ascertaining D. carota reproduction-goal to improved sugariness or sugar making (Alasalvar et al., 2001). Though saccharides are not only in forming or build deviations in the sugari-ness of uncooked D. carota, privileged sugar contents and enhanced sweetness are advantageous aspects for budding and humanizing D. carota qualities ( Alasalvar, et al., 2001; Simon et al., 1980). The developed method using HPLC with refractive index proved to be a swift and proficient technique for quantification and identification of individual sugars. Sample preparation was easy and purified water was used as mobile phase, produced nonhazardous scum. All validation steps proved that the developed method is efficient state of art. DCP cultivar forms utmost sweetie point as contrast to new verities. For that reason, DCP cultivar might be utilized in position of further D. carota multiplicities in regulate to get benefit of its nutra-ceuti-cal workings.

Table 4.6 Linear-regression. parameters attained from sugar values calibration curves.

Sugars Regression-equation R2 R2adj p-value*

Fructose y = 173,598.74. x - 9,523.21 0.999 0.999 0.22

Glucose y = 235,346.62. x - 2,056.31 0.999 0.996 0.14

Maltose y = 234,318.23. x - 9,572.42 0.999 0.998 0.12

Maltotriose y = 235,732.16. x - 12,368.792 0.999 0.996 0.13

Maltodextrin y = 234,731.16. x - 12,388.799 0.999 0.998 0.15

2 2 Where: R. adj: adjusted regression-coefficient R. : regression-coefficient, and *Probabilityvalues

Table 4.7 Validation parameters for the liquid chromatography method

Repeatability (intra-day) Intermediate tR LOQ LOD Sugar Accuracy (min) mg/L mg/L (Inter-day) DCW DCY DCP T29 DCR DC3 DC90

Fructose 13.0 0.32 0.56 0.63 0.45 0.39 0.38 0.61 1.82 55.63 62.32

Glucose 12.1 0.58 0.24 0.45 0.31 0.37 0.27 0.32 0.31 178.23 59.45

Maltose 9.3 0.28 0.39 0.51 0.36 0.56 0.31 0.45 0.69 165.33 48.13

Maltotriose 8.1 0.53 0.41 0.91 0.61 0.45 0.51 0.56 0.72 142.32 56.52

Maltodextrin 6.8 0.43 0.34 0.45 0.31 0.51 0.41 0.98 2.55 49.36 59.35

Results of repeatability and intermediate accuracy expressed as percentage CV (%). LOQ: limit of quantification; LOD: limit of detection.

Table 4.8 Sugar recovery rates (standards) added to the D. carota cultivars samples. Recovery rate of sugars standards (%) Fructose Glucose Maltose Maltotriose Maltodextrin aStandard 1

DCW 88.99 88.91 88.29 90.23 89.64 DCY 88.34 89.98 86.09 91.45 90.55 DCP 90.85 84.33 85.89 90.39 91.56

T29 86.48 86.89 88.91 91.78 90.05 DCR 87.50 85.98 89.88 92.42 88.98 DC3 88.56 86.36 87.73 91.83 90.91 DC90 86.67 87.98 88.45 90.25 90.00 bStandard 2

DCW 96.99 92.88 98.89 97.88 97.88 DCY 95.85 91.66 97.85 98.08 98.88

DCP 94.85 94.88 98.80 98.04 98.87

T29 97.45 93.05 98.86 97.78 98.05 DCR 95.39 90.77 98.88 97.89 97.00 DC3 93.78 91.67 98.73 97.98 98.76 DC90 93.80 97.66 97.89 97.99 97.67 cStandard 3

DCW 99.00 97.88 98.07 98.81 98.88 DCY 98.85 98.98 97.85 98.56 98.56 DCP 97.85 98.80 96.89 98.44 97.87 T29 98.48 98.45 98.86 97.78 98.05 DCR 98.55 97.98 98.88 98.89 97.98 DC3 98.66 97.77 97.73 97.98 98.75 DC90 97.56 97.56 97.45 98.09 97.98

Continue Table 4.5 dStandard 4 DCW 99.34 97.88 98.29 98.48 99.78 DCY 98.85 98.98 99.09 98.56 98.98 DCP 97.85 99.45 96.89 99.49 98.16 T29 98.48 98.45 99.91 97.78 98.05

DCR 99.50 97.98 98.88 98.89 97.98 DC3 98.67 99.89 97.73 97.87 98.97 DC90 97.59 97.98 98.45 98.88 99.79 eStandard 5

DCW 98.85 97.88 98.85 100.0 97.88

DCY 97.85 100.0 97.85 98.08 98.88 DCP 98.80 98.04 98.80 98.04 98.87

T29 100.0 98.05 98.86 97.78 98.05 DCR 98.88 97.89 99.88 97.89 100.0 DC3 98.78 100.0 98.70 97.98 98.76 DC90 98.83 97.98 99.99 97.99 97.67 aStandard1 {Fructose (0.25), Glucose (0.50), Maltose (0.60), Matotriose (0.80), Maltodextrin (1.00)}, bStandard2 {Fructose (1.25), Glucose (1.50), Maltose (1.60), Matotriose (1.80), Maltodextrin (2.00)},cStandard3 {Fructose (2.25), Glucose (3.50), Maltose (3.60), Matotriose (3.80), Maltodextrin (5.00)},dStandard 4 {Fructose (4.25), Glucose (4.50), Maltose (4.60), Matotriose (4.80), Maltodextrin (1.00)}&eStandard 5 {Fructose (5.25), Glucose (5.50), Maltose (5.60), Matotriose (5.80), Maltodextrin (6.00)}. Weights of standards are in g/100mL.

Table 4.9 -Concentration (means- ± -standard-deviation) of sugars-in-different-cultivars-of-D. carota.

Individual Sugars (g/100g) D. carota cultivars Fructose-. Glucose-. Maltose-. Matotriose-. Maltodextrin-. Total sugars-.

d c e a b f 1.103a ±0.001 0.963a ±0.001 1.986b ±0.002 0.048e ±0.001 0.411f ±0.001 4.511b ±0.015

(23.44%) (21.80%) (44.79%) (1.01%) (8.96%) (100%) DCW

d c e a b f 1.351e ±0.002 1.127e ±0.002 2.463g ±0.004 0.058g ±0.002 0.166a ±0.001 5.165e ±0.058 DCY (25.53%) (22.03%) (48.07%) (1.13%) (3.24%) (100%)

d c e a b f 2.090g ±0.004 1.395g ±0.003 2.194f ±0.003 0.049f ±0.001 0.583g ±0.003 6.311g ±0.034 DCP (32.25%) (22.40%) (35.21%) (0.79%) (9.35%) (100%)

d c e a b f 1.442f ±0.002 1.311f ±0.003 2.148c ±0.002 0.029c ±0.002 0.351d ±0.001 5.281f ±0.041 T29 (27.10%) (25.40%) (40.37%) (0.54%) (6.59%) (100%)

d c e a b f 1.272c ±0.003 1.026c ±0.002 2.441e ±0.001 0.004a ±0.001 0.169b ±0.002 4.912c ±0.011 DCR (25.90%) (20.89%) (49.70%) (0.08%) (3.43%) (100%)

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Continue Table 4.9 d c e a b f 1.303d ±0.001 0.987b ±0.001 2.365d ±0.003 0.039d ±0.002 0.405e ±0.003 5.099d ±0.026 DC3 (25.65%) (19.44%) (46.55%) (0.76%) (7.60%) (100%)

d c e a b f 1.205b ±0.002 1.043d ±0.002 1.886a ±0.001 0.019b ±0.001 0.335c ±0.002 4.488a ±0.018 DC90 (26.83%) (23.23%) (42.02%) (0.42%) (7.50%) (100%)

The values in ( ) showed the percentage composition of sugar types. Superscript letters represent significance-difference (p < 0.05) amongst the sugar types in each cultivar (row wise). Subscript letters represent the significance-difference (p < 0.05)amongst cultivars (column wise)

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Figure 4.1Chromatogram of sugars profile for test-mixture of sugars-standards. Peak identification (1-) maltodextrin, (2-) maltotriose, (3-) maltose, (4-) glucose and (5-) fructose

102

Figure 4.2 Typi-cal-chromatograms of sugarprofiles for DCP cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Figure 4.3Typical chromatograms of sugar profiles for DC90 cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Figure 4.4Typical chromatograms of sugar profiles for DC3 cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Figure 4.5Typical chromatograms of sugar profiles for DCW cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

106

Figure 4.6Typical chromatograms of sugar profiles for DCY cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Figure 4.7Typical chromatograms of sugar profiles for T29 cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Figure 4.8 Typical chromatograms of sugar profiles for DCW cultivar. Peak identification (1) maltodextrin, (2) maltotriose, (3) maltose, (4) glucose and (5) fructose

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Calibration-curve for individual maltodextrin standard at working conditions 5000000

4500000 y = 234,731.16. x - 12,388.799 R² = 0.999 4000000

3500000

3000000

2500000

2000000 Peak Peak area (µV)

1500000

1000000

500000

0 0 1 2 3 4 5 6 Concentration (g/100mL)

Figure 4.9 Calibration-curve for maltodextrin (individual) at operational conditions (standard)

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Calibration-curve for individual maltotriose standard at working conditions 6000000

y = 235,732.16. x - 12,368.792 5000000 R² = 0.999

4000000

3000000 Peak area (µV) 2000000

1000000

0 0 1 2 3 4 5 6 Concentration (g/100mL)

Figure 4.10 Calibration-curve for maltotriose (individual) at operational conditions (standard)

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Calibration-curve for individual maltose standard at working conditions 7000000 y = 234,318.23. x - 9,572.42 R² = 0.999

6000000

5000000

4000000

3000000 Peak Peak area (µV)

2000000

1000000

0 0 1 2 3 4 5 6 Concentration (g/100mL)

Figure 4.11 Calibration-curve for maltose (individual) at operational conditions (standard)

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Calibration-curve for individual glucose standard at working conditions 30000000 y = 235,346.62. x - 2,056.31 R² = 0.999 25000000

20000000

15000000 Peak Peak area (µV) 10000000

5000000

0 0 1 2 3 4 5 6 Concentration (g/100mL)

Figure 4.12 Calibration-curve for glucose (individual) at operational conditions (standard)

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Calibration-curve for individual fructose standard at working conditions 18000000 y = 173,598.74. x - 9,523.21 R² = 0.999 16000000

14000000

12000000

10000000

8000000 Peak Peak area (µV) 6000000

4000000

2000000

0 0 1 2 3 4 5 6 Concentration (g/100mL)

Figure 4.13 Calibration-curve for fructose (individual) at operational conditions (standard)

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4.2.2 Quantification of individual phenolic compounds in different cultivars of D. carota roots Phenlolic acids are commonly found in vegetable-foods as nonessential dietary-components, generallyrecognized as plants secondary-metabolites (Faisal et al., 2016). These are anti- nutritive owing to the undesirable effects on digestibility of proteins. In addition to other qualities these are essentialowing to their inhibition againstmalignant cellsand (Martinez- Valverde, 2000). Figures 4.14 – 4.21 showed the typical chromatograms of phenolic compounds extracted from selected cultivars of D. carota. All the cultivars contained

derivatives of hydroxycinnamic-acid i.e. 3,4--””diferuloyl-quinic-acerbic, ”cis-3-‘ -”caffeoyl-

quinic-”acid, 5--‘” caffeoyl-quinic-acid, Caffe-ic” -acid, 3,5--”” dicaffeoyl-quinic-acid, 3 ” -p—

coumaroyl-quinic” -acid, 3 ”—feruloy-quinic ” -acid, 3,”4--” dicaffeoyl-quinic-acid, ”5 ” --feruloy-

quinic-acid, 3--” caffeoyl-quinic-acid,, ” cis-5--” caffeoyl-quinic-acid, ”5--p-” coumaroyl-quinic-

acid, ”4--” feruloy-quinic-acid, and 3, ”5--” diferuloyl-quinic-acid.These derivatives were

recognized and computed by evaluating retention times and peak areas with standards. ’ Table 4.10 revealed the quantitative values of total detectable and individual phenolic

compounds. 5-’caffeoylquinic acid was the most important’ hydroxylcinnamic’ -acid present in all the cultivars, varied from 6.98-33.23mg/100g of total phenolic-compounds. The total amount of phenolic compounds in DCP cultivar was 54.62 mg/100g FW, where as the corresponding values in other cultivars ranged from 12.8 to 20.29 mg/100g FW.

Higher contents (ρ < 0.05) of 3-’caffeoyl-quinic-acid, cis-3-caffeoyl-quinic-acid, 5--

caffeoyl-quinic-acid, Caffe-ic-acid, 3 ’-p--coumaroyl-quinic-acid, 3--’ feruloy-quinic-acid, 5--

’feruloy-quinic-acid, cis-5 ’—caffeoyl-quinic-acid, 5 ’ -p—coumaroyl-quinic-acid, 4--’ feruloy-

quinic-acid and 3,5--’ diferuloyl-quinic-acid was the main phenolic compounds identified in

DCP cultivar. The compounds 3,4-’ dicaffeoylquinic acid, 3,5-’’ dicaffeoylquinic acid and 3,4-

’diferuloylquinic acid were the major phenolic-acids present in DC3, T29 and DCW cultivars.

DCP cultivar was the only one which contained Caffeic ’ acid (1.93 mg/100g FW) among all the selected cultivars, which is comparable to the result (2.42 mg/100g FW) reported by Alasalvar et al. (2001) in different colored carrots. In the current cram various phenolic-composites were not perceived like iso-

coumarins 6-’hydroxymelle and 6--’ meth-oxy-mellein as reported by Harding et al. (1980) and P-hydro-xyl-benz-oic-acid as investigated by Babic et al. (1993). In total 14 types of phenolic acids were detected in the present work but Alasalvar et al. (2001) investigated that nine to

elevenhydroxycinnamic’ derivatives were found in carrots cultivars having different

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colors.The variation in phenolic-acid composition may differ between cultivars, as well as among parts within the individual plant (Francisco et al., 2010). Sing et al. (2001) cited the existence of unknown kinds of phenolic composites in carrot cultivars. The presence of unknown phenolic compounds may depend upon the root colour or location of the cultivation region (Bajaj et al., 1980). On the other hand genetic back ground also an authentic factor which could affect the quantitative chemical composition of plant tissues (White & Broadley, 2005). The variation in phenolic composition among the carrot cultivars of present study is also due to the difference in cultivars having variable color attributes. The data regarding the phenolic composition revealed that carrot cultivars native to Pakistan proved to be potential sources of antioxidants, bioactive compounds. Chemometric tools were used to evaluate cultivars with higher amount of individual and total amount of phenolic acids. Purple colour carrots (DCP cultivar) were found to be potential source of phenolic acids whereas DCY showed lowest concentration among the selected cultivars. Identification of phenolic rich cultivars could potentially increase the intake of health promoting attributes through diet and helpful in the anticipation of chronic diseases in human beings. Cultivars with high concentration may be used in process industries for the preparation of functional foods. These may also be used for the preparation of anti-cancer and anti-aging medicines/creams on commercial scales after extraction and purification of phenolic compounds.

4.2.3 Quantification of β-carotene β-carotene is recognized for many health benefits like anti-cancer potency, anti-ageing effects, visibility health booster and defensive against cardiovascular diseases (Breithaupt & Bamedi, 2001). Generally β-carotene is used as oral sun protectant but studies showed that it can also be used against light induced erythema (Stahl et al., 2000). It is very important to explore the newly invented cultivars of health promoting vegetables regarding their functional ingredients. The food processing can changes matrix structure in positive (increase of certain ingredients) and negative (loss of certain ingredients) ways (Livny et al., 2003). In view of that determination of best conditions and solvents for the extraction of required constituents with high yield is very important (Złotek et al., 2016).

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Table 4.10 Pheno-lic-acids-profile in different-cultivars of D. carota (carrot) roots

Composition (mg/100g of fresh weight )

Peak #h Phenolic Compound T-29 DC-R DC-3 DC-90 DC-Y DC-P DC-W

c f d e g a b 1 3--”” caffeoyl-quinic-acid ” 0.320±0.02 0.220±0.01 0.28±0.02 0.24±0.01 0.07±0.00 0.92±0.04 0.82±0.03

b a 2 cis-”3 ” —caffeoy-lquinic-acid” ND ND ND 0.08±0.01 ND 2.11±0.01 ND

e d b c f a g 3 5--” caffeoyl-quinic-acid” 10.20±0.30 9.120±0.38 11.32±0.42 9.91±0.31 8.89±0.33 33.23±0.42 6.98±0.28

4 ”Caffe-ic ” -acid” ND ND ND ND ND 1.93±0.13 ND

b e d c g a f 5 3-p--” coumaroyl-quinic-acid ” 0.630±0.01 0.550±0.02 0.58±0.02 0.61±0.02 0.12±0.01 0.81±0.10 0.14±0.01

c b e d f a g 6 3--”” feruloy-quinic-acid” 0.31±0.01 0.32±0.02 0.24±0.02 0.31±0.02 0.16±0.01 8.83±0.04 0.09±0.00

c d a e f c b 7 3,4--”” dicaffeoyl-quinic-acid ” 3.12±0.03 2.9±0.02 3.62±0.03 2.41±0.02 1.90±0.02 3.12±0.03 3.33±0.03

d e f c g a b 8 5--”” feruloy-quinic-acid” 0.21±0.01 0.15±0.01 0.11±.01 0.26±0.01 0.06±0.01 0.83±0.02 0.72±0.02

a b 9 cis-” ”5—caffeoyl-quinic-acid” ND ND ND ND ND 0.31±0.01 0.05±0.00

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Continue Table 4.10

d c b e f a 10 5—p-”- ” coumaroyl-quinic-acid 0.10±0.01 0.11±0.01 0.16±0.02 0.09±0.00 0.06±0.00 0.79±0.00 ND

a b 11 4-” - ” feruloy-quinic acid 0.51±0.03 ND ND ND ND 0.43±0.02 ND

a c b d e f 12 3,5 ”- ” dicaffeoylquinic-acid 4.2±0.04 3.5±0.03 3.8±0.03 1.56±0.02 1.49±0.02 0.19±0.01 ND

d f e b c a 13 3,4--”” diferuloyl-quinic-acid 0.11±0.01 0.08±0.00 0.07±0.00 3.25±0.02 ND 0.43±0.02 3.96±0.03

b c e a d 14 3,5--”” diferuloyl-quinic-acid ND 0.12±0.01 0.11±0.01 ND 0.05±0.00 0.69±0.01 0.06±0.00

Total Phenolic 19.71±0.08c 17.07±0.10e 20.29±0.08b 18.72±0.09d 12.8±0.12g 54.62±1.11a 16.15±0.09f

The values are expressed in a-fmean ± standard deviation followed by the same letter, within a row, are not significantly different (ρ > 0.05).hPeak numbers corresponds to the peaks in Figure 2. ND mean not detected.

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Figure 4.14 Typical-HPLC-chromatograph blend of standard [typical] phenolics. Peak recognition [1] 3--” caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-

quinic-acid, [3] ”5”-”- caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-

”feruloyquinic acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13]

3,4--”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.15 Typical-HPLC-chromatograph of phenolic acids in T29 cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-quinic-

acid, [3] ”5”-”-caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4” -dicaffeoylquinic acid,[8] 5-”feruloyquinic

acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13] 3,4--

”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure4.16 Typical-HPLC-chromatograph of phenolic acids in DCY cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-

quinic-acid, [3] ”5”-”- caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-

”feruloyquinic acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13]

3,4--”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.17 Typical-HPLC-chromatograph of phenolic acids in DCR cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-

quinic-acid, [3] ”5”-”- caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-

”feruloyquinic acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13]

3,4--”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.18 Typical-HPLC-chromatograph of phenolic acids in DC90 cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-

quinic-acid, [3] ”5”-”- caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-

”feruloyquinic acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13]

3,4--”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.19 Typical-HPLC-chromatograph of phenolic acids in DCP cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-quinic-

acid, [3] ”5”-”-caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4” -dicaffeoylquinic acid,[8] 5-”feruloyquinic

acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13] 3,4--

”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.20 Typical-HPLC-chromatograph of acids in DCW cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-quinic-acid,

[3] ”5”-”-caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-”” feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-” feruloyquinic

acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13] 3,4--

”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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Figure 4.21 Typical-HPLC-chromatograph of phenolic acids in DC3 cultivar. Peak recognition [1] 3--”caffeoyl-quinic-acid, ”[2] cis-3--”caffeoyl-

quinic-acid, [3] ”5”-”- caffeoylquinic acid,[4] ”caffeic acid,[5]3-”p-coumaroylquinic acid, [6]3-””feruloyquinic acid, [7] 3,4”-dicaffeoylquinic acid,[8] 5-

”feruloyquinic acid,[9] cis-”5—caffeoyl-quinic-acid, [10] 5--p-”counaroyl-quinic-acid, [11] 4--”feruloy-quinic-acid, [12] 3,5--”dicaffeoyl-quinic-acid, [13]

3,4--”diferuloyl-qunic-acid and [14] 3,5--”diferuloy-quinic acid

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In the present study yield of β-carotenes from selected cultivars was investigated by using different solvents and findings are presented in Table 4.8. Highest yield of β-carotene (14.69mg/100g) was extracted from DCP cultivar by 2-propanol at 60°C and that of lowest (3.78mg/100g) from DCW by ethanol at 25°C. Statistical analysis presented that β-carotene counts assorted considerably (ρ < 0.05) between the cultivars and extraction conditions. It can be claimed that high temperature is more suitable for the extraction of β-carotene and there is a possibility that the bounded form of carotene was not released at low temperature condition and presented the poor extraction yield. The earlier studies are also seemed to be claiming that higher temperature could release β-carotene bounded with protein and easily extractable (Dutta et al., 2005). The results regarding the β-carotene yield are not comparable to the previously cited findings (2.47 to 6.45mg/100g) in carrots extracted at different conditions of temperature and solvent (Fikselova et al., 2008), whereas most of the cultivars showed the comparable β- carotene contents to that (6.2 to 11.6 mg/100g) in carrots presented in another study (Wingqvist, 2011). The difference and variation of findings regarding β-carotene contents may be due to difference in cultivar, extraction conditions and type of solvent. It is very notable that DCP cultivar was much better in β-carotene content at high temperature extraction. However, some cultivars showed very poor response in β-carotene contents and the reason might be the low temperature employed for extraction or the genetic makeup of new lines. In view of data regarding β-carotene contents in selected cultivars of D. carota it may be concluded that cultivars of the present study are the potent source of β-carotene. The increase in temperature (up to 60°C) influenced the extraction yield of β-carotene in 2- prpanol positively. These temperature conditions and solvent can be recommended for extraction technology of carrot processing. The described method for carotenoids extraction can be modified by applying different temperature conditions, changing solvents and timings. Validation of results of this method proved that objective might be scale-up to industrial applications. The DCP showing good β-carotene contents among the selected cultivars of present study may be cultivated on large to attain high production of β-carotene, which may be helpful to overcome the shortage of value added products having β-carotene as a major ingredient. Such types of cultivars could find many applications in food supplementation and fortification.

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Table 4.11 β-carotene yield (mg/100g) extracted from D. carota cultivars at different temperatures At 25°C At 60 °C At 25°C At 60°C Carrot Cultivars with ethanol* with ethanol* with 2-propanol* with 2-propanol*

d b c a DCW 3.78±’0.09 5.22±’0.13 4.84±’0.05 6.22±’0.10

d b c a DCY 4.57±’0.08 6.21±’0.14 5.44±’0.04 7.21±’0.14

d c b a DCP 9.31±0.10 10.69±’0.15 11.13±’0.13 14.69±’0.18

d c b a T29 5.11±’0.07 7.33 ± ’0.11 7.45 ± ’0.06 9.33±’0.11

d c b a DCR 4.41±’0.05 6.96±’0.08 7.11±’0.08 8.96±’0.14

d c b a DC3 5.53±’0.07 6.14±’0.09 7.94±’0.09 9.96±’0.14

d c b a DC90 5.11±’0.06 8.25±’0.10 8.97±’0.05 10.25±’0.12

*The values are expressed in mean ± standard deviation. The superscript alphabets in the row show significant difference (ρ < 0.05).

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Part III 4.3 Antioxidant activities 4.3.1 Antioxidant activities of D. carota roots 4.3.1.1 Total phenolics (TP) Total phenolic-acid contents (TP) in extracts of selected cultivars of D. carota were determined byFolin Ciocalteu-assay. The findings regarding the TP contents in selected cultivars of D. carota are presented in Table 4.9. The Highest TP contents (65.39 mg/100g FW) were found in DCP and that of lowest (30.26mg/100g FW) in DCY cultivar. Statistical analysis of data showed that TP values diverse considerably (ρ < 0.05) between cultivars. Kahkonen et al. (1999) reportedgallic-acid as phenolic-acid equivalents in D. carota-bark and dermis, which consisted of6.60 & 0.60mg/g respectively. Vinsonn et al. (1998) cited that 46.40mg catechin-equivalents were found in 100g of fresh carrots. The variation of results in the previously published studies may be due to changed extraction-methods and the units to present the findings. Phenolic-acids contents among novel cultivars of D. carota were determined by using two different methods i.e HPLC andFolin Ciocalteu-assay, which showed thatphonic-acid contents turn-down in the similar-order. Nevertheless, phenolics were on higher side when determined by Folin Ciocalteu-assay, as compare to the findings evaluated by HPLC in D. carota cultivars. Howard et al. (2000) found the same trends in results, among both analytical-approaches. This might be probable owing to the unnecessaryinformative of insignificant phenolic acids or other reducing compounds (superfluous) persistent in sample- extracts from each cultivar or origin. Remarkable characteristics of D. carota are sufficient to prove that it might be use infast-food servings and prepared-salads. It may be endorsed that the phenolic acid contents in higher concentration are found in the DCP cultivar, which might be used in the preparation of supplemented-diets (having phenolic acids) in process industries on commercial scales. 4.3.1.2 Total ascorbic acid (TAA) High blood pressure, heart diseases and endothelial-dysfunction might be cured by ascorbic acid. Dynamic growth in plants is regulated by ascorbic-acid and found in variable amounts among cultivars or species (Lee et al., 2000). Vitamin C is effective against scurvy and reduces the risk of cold on human body (Center, 2003). Table 4.9 presented TAA contentsin extracts form respective cultivars of D. carota. TAA levels varied from 41.12- 58.36 mg/100 g of FW (ρ < 0.05) which were comparable to the result (54.5 mg/100g FW) in red carrots

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(Venkatachalam et al., 2014). Some of the cultivars of present study have lower concentration of TAA like DCW (43.29 mg/100g FW), DCY (41.12 mg/100g FW), T29 (48.56 mg/100g FW), DCR (46.39 mg/100g ) and DC90 (47.39 mg/100g ). The difference of TAA values in literature may be due to the difference in genotype or methods of determination.bin comparison to other D. carotacultivars; DC3 and DCPfound to have higher TAA contents and that of lowest were found in DCW and DCY cultivars. Temperature variations and light intensity are two main parameters which regulates the production of ascorbic acid. (Harris, 1975). Its proposed function has been reported in photosynthesis as enzyme-cofactor and cell growth controller (Smirnoff & Wheeler, 2000). It has been cited that vitamin-C is very unstable at higher temperature (Igwemmar et al., 2013); they studied the effect of heat on vitamin-C in different vegetables and reported that overheating and excess water results in depletion ascorbic acid. It may be concluded that vegetables and fruits in summer season has more vitamin-C as reported in carrot and oranges (Plaza et al., 2006). In view of discussion about ascorbic acid it may be concluded that selected cultivars of D. carota are the potent source of vitamin-C. These may be used in the production of value added products on commercial scales to cure diseases cause by the lacked of vitamin-C. 4.3.1.3 Total antioxidant capacity (TAC) TAC values obtained from respective D. carota cultivars are presented in Table 4.10, which showed that TAC values varied from 41.56-77.69 mg/100 g. The statistical data regarding the TAC revealed that TAC values diverse considerably (ρ < 0.05) between the selected cultivars of D. carota. Higher contents of TAC (77.69 mg/100g) were found in DCP cultivar, which is not comparable to the TAC results cited by Venkatachalam et al. (2014), reporting 47.65±1.20 mg/100gin red carrots only.But it is in agreement with TAC results found in DCR, T29, and DC90 cultivars obtained in currentwork. It has been reported that TAC results go on higher side with an increase in contents of ascorbic and phenolic acid (Faisal et al., 2016). The poly-phenolic contentsplay vital role in superlative multifunctionality of anti-oxidants in nature (Slusarczyk et al., 2009). Several studies in literature are available which reviewed the presence antioxidant contents inD. carota. The difference of TAC values in these studies may be due to the difference in genotype or methods used for the determination of TAC. In view of the findings related to total antioxidant activity in selected cultivars of D. carota it may be concluded that selected cultivars are potent source of antioxidants like phenolic acids, carotenoids compounds and ascorbic acid. It could be recommended that these cultivars having potential against oxidation process can be used in the synthesis of functional foods and medicines to cure many diseases caused by oxidation process in body.

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4.3.1.4 Free radical scavenging capacities Findings regarding the DPPH scavenging capacity are presented in Tables 4.10, which revealed that DPPH values varied from 27.55-52.36%. The findings regarding DPPH capacity of present study are not comparable to Zhang et al. (2004). They reported DPPH scavenging capacity in carrot-peel (75.8%). The difference in findings of current study is due the difference in sample preparation .i.e. full carrot root was used in extraction process. A decreasing trend was observed in DPPH scavenging capacity of selected cultivars of carrot due the presence of variable amount of phenolic acids or other anti-oxidants, having different responses to testing methods. The Increasing trend of DPPH scavenging-activity was found

in selected cultivar i.e DC-P‘‘< DC-3 <> T-29‘< DC-90‘< DC-R‘< DC-W‘< DC-Y‘. ‘ Table 4.10 explained to super-oxide-radical sca-ven-ging-activity in chosen cultivars of D. carota, varied from 26.550-53.690 mg/100g. Along with DC-W, DC-Y, DC-P, T-29, DC-R, DC-3 and DC-90 cultivars, the DC-P confirm higher super-oxide-radical-sca-ven-ging capacity with 53.690mg/100g (ρ < 0.05). Maheswari et al. (2012) and Lu et al. (2011) described that generally poly-phenols throw in to anti-oxidant capacities of variety of fruits & vegetables. During current examine the higher rate of super-oxide-radical sca-ven-ging capacity in DC-P cultivar is owing to occurrence of lofty values of phenolic-acids and its derivative forms. Table 4.10 explained to hydroxyl –radical-sca-ven-ging capacity of the chosen cultivars of carrot, varied from 28.120-51.910 mg/100 g (ρ < 0.05). The tendency was similar

as to of DPPH sca-ven-ging-activity i.e. DC-P‘‘< DC-3 <> T-29‘< DC-90‘< DC-R‘< DC-W‘< DC- Y. Though, the anti-oxidant-capacity of chosen fruits & vegetables in this research (Table 3) was linked through their phenolics (Table 1). It was in concurrence with earlier available outcomes described by Alasalvar et al. (2001). 4.3.1.4 Liaison between phenolic content and antioxidant properties in Daucus carota Phenolic contents are the indicators of antioxidant properties of fruit and vegetables (Piluzza & Bullitta, 2011). In the present study different types of assays has been conducted to evaluate the biological activities of selected cultivars of D. carota. Figure 4.22 and 4.23 showed a well linear correlation in-between the phenolic acid contents and antioxidant capacities. The data regarding antioxidant activities showed that DCP cultivar showed highest value of antioxidant activity, having high amount of phenolic compounds and that of lowest by DCY cultivar, having lowest amount of Phenolic compounds. The same ratio was

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observed in case of other cultivars. The results regarding phenolic acid contents and antioxidant activity were in agreement with the findings described by Lee et al. (1995) in which they reported that rectilinear-relationship was found in anti-oxidant activity and phenolics contents in capsicum. Much work has been cited regarding structure activity relationship of phenolic acids and it has been found that poly-phenolic acids are more efficient that mono-phenolic acids (Shahidi, 1997). In the present study carrot cultivars have considerable concentration of phenolic acids, which showed remarkable antioxidant activities. DCP cultivars have higher concentration of phenolic acids among selected cultivars having high antioxidant activity. No considerable-correspondence was established after having bulk data about phenolics and antioxidant activity, which may be due to presence of variable amounts of antioxidants. Including carotenes, victual-vitamins, bioflavonoids (Zhang & Hamauzu, 2004), effectingcapacity of antioxidants. Afterwards, antioxidant capacity due to phenolic-acids is effected due the presence of ascorbic-acid and carotenoids compounds 4.3.2 Antioxidant activities of dehydrated D. carota top whole 4.3.2.1 Total phenolics (TP) The oxidative reactions are the main reason of food deterioration. Such reactions not only results in the loss of nutritional values of fruits and vegetables but also cause decline in taste, aroma and texture (Lund et al., 2011). The phenolic acids have the ability to stop or minimize the oxidative reactions (Laguerre et al., 2007). The data regarding the total phenolic acid contents in top whole of selected cultivars of D. carota is presented in Table 4.14. The total pheonlic acid contents varied from 6.13mg/g for DCY to 22.15 for DCP cultivar. Statistical analysis regarding total phenolic acid contents showed significant (ρ < 0.05) differences among top whole of D. carota cultivars. The findings regarding the total phenolic acid contents in top whole of selected cultivars are not comparable with previously reported results (7.2 mg/GAEg dw) in carrot leaves. The difference in result is may be due to difference in plant material under study. The highest phenolic acid contents (22.15) were found in top whole of DCP cultivar. The trend of high phenolic acid contents in top whole of DCP cultivar is in agreement with previously reported results (58.36mg/100g) extracted from root part of DCP cultivar (Faisal et al., 2016). In view of findings related to total pheolic acid contents it may be concluded that selected cultivars of D. carota have significant amount (highest in DCP) of total phenolic acid contents, having medicated properties, which may as medicinal plant.

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Table 4.12 Total ascor-bic-acid & phenolic-acids of selected (chosen) varities of D. carota (carrot) roots

Total, phenolic-acids (mg,/100g)* by spectro- D. carota cultivars Total, ascorbic-acids (mg,/100g)* photo-meter

b b DCW 43.29±’0.32 32.85 ±’0.23 ’

a a DCY 41.12±’0.22 30.26 ±’0.31

g g DCP 58.36±’1.46 65.39 ±’1.81

e e T29 48.56±’0.92 36.92±’0.26

c c DCR 46.39±’0.34 33.21±’0.21

f f DC3 51.21±’0.49 42.64±’0.20

d d DC90 47.39±’0.38 38.56±’0.38

*The Findings are articulated in mean ± standard divergence. The super-script alpha-bets with in column illustrate noteworthy difference (ρ < 0.05).

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Table 4.13 Anti-oxi-dant-capacitie of chosen varities of D. carota (carrot) roots scavenging activity scavenging Antio-xidant activity scavenging activity Extracts of D. (DPPH) activity(Hydroxyl radical) (Total) (Superoxide radical) carota cultivars (mg/100g-FW)* (mg/100g FW )* (mg/100g-FW)* (mg/100g FW)*

b b b b 28.560 ‘±’0.150 28.230 ’±0.140 43.260 ‘±’0.210 29.250 ‘’±0.110 ‘D ‘CW

a a a a 27.550 ‘±’0.120 28.120 ‘±0.110 41.560 ‘±’0.180 26.550 ‘’±0.130 ‘D ‘CY

g g g g 52.360 0 ‘±’0.760 51.910 ‘’±0.630 77.690 ‘±’1.120 53.690 ‘’±0.820 ‘D ‘CP

e e e e 32.810 ‘±’0.090 38.360 ’±0.110 53.120 ‘±’0.450 35.390 ‘’±0.120 ‘T ‘29

d d d d 29.890 ‘±’0.110 33.320 ‘’±0.090 45.600 ‘±’0.350 30.120 ‘’±0.100 ‘D ‘CR

f f f f 38.360 ‘’±0.120 35.120 ‘’±0.140 55.530 ‘±’0.320 36.930 ‘’±0.130 D ‘C3

c c c c 31.330 ‘±’0.130 32.510 ‘’±0.160 45.350 ‘±’0.340 29.630 ’ ‘±0110 D ‘C90

*The findings are articulated mean ± standard divergence. The supe-rscript alphabets in columns illustrate noteworthy dissimilarity (ρ < 0.05).

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Scatter plot of total phenolic acid contents and total antioxidant activity 90

Total phenloic acid contentTotal (mg/100g) 10

0 0 10 20 30 40 50 60 70 Total antioxidant activity

Figure 4.22 Relationship between total antioxidant activity and total phenolic contents in D. carota

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Scatter plot of total phenolic acid contents and DPPH scavenging capacity 60

10 Total phenloic acid contentTotal (mg/100g)

0 0 10 20 30 40 50 60 70 DPPH scavenging capacity

Figure 4.23 Relationship between DPPH scavenging activity with total phenolic contents in D. carota.

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4.3.2.2 Total antioxidant capacity (TAC) The principal function of antioxidants is the inhibition the initiation of oxidation, which results in delaying of oxidation process (Namiki, 1990; Valko et al., 2007). The data regarding the total antioxidant capacity (TAC) of dehydrated extract from top whole of selected cultivars of D. carota are presented in Table 4.14. It showed that lowest TAA (40.41mg/100g) was found in DCY and that of highest (73.51mg/100g) in top whole of DCP cultivar. Statistical analysis revealed that TAC in selected cultivars varied significantly (ρ < 0.05) among top whole of D. carota cultivars. The findings regarding the TAC in DCP cultivar is comparable to TAC (66.4mg/100g) of spinach (Ismail et al., 2004) and not comparable {47.0 µsM Fe (II)/g} to fenugreek leaves extract (Premanath et al., 2011). On the other hand TAC of fenugreek leaves extract was quite comparable to the top whole of other cultivars of present study. Form findings regarding TAC, it may be concluded that top whole of selected cultivars of D. carota has significant TAC values comparable to other leafy vegetables. 4.3.2.3 Free radical scavenging capacities Animals have defensive system against oxidation in order to protect cells against excessive level of free radicals (Bouayed & Bohn, 2010). Absorption of compounds such as vitamins, minerals and protein give additional protection when consume with daily diet (Ostrovidov et al., 2000; Divisi et al., 2006). These antioxidant compounds have ability to neutralize free radicals (Slemmer et al., 2008). In view of the importance of an anti-oxidative nature of leafy vegetables following assay about free radical scavenging capacities of top whole of selected cultivars of D. carota has been studied which are given below. The data about DPPH scavenging capacity of top whole of selected cultivars of D, carota is presented in Table 4.14. It showed that the lowest DPPH (24.55mg/100g) was found in DCY cultivar and that of highest (49.81mg/100g) in top whole of DCP cultivar. Statistical analysis revealed that DPPH scavenging capacities in selected cultivars varied significantly (ρ < 0.05) among top whole of D. carota cultivars. The findings of present study about DPPH capacity is not in agreement with (1.75 - 1801.56 μg/mL) other conventional vegetables (Chao et al., 2014). The difference in DPPH capacities is may be due to difference in vegeTable type having different concentration of phenolic acids or other antioxidant compounds.

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Free radicals are highly reactive in nature; attack on macromolecules which results in cell damage and disruption. Target of these radicals includes nucleic acids, lipids and proteins (Lobo et al., 2010). The data regarding superoxide radical capacity of top whole of D. carota cultivars are presented in Table 4.14. It showed that superoxide radical capacity values ranged from 25.23mg/100g for DCY to 49.82mg/100g for DCP cultivar. Statistical analysis revealed that superoxide radical capacities in selected cultivars varied significantly (ρ < 0.05) among top whole of D. carota cultivars. Findings regarding superoxide radical capacity are impossible to compare with previously published result (31.39%) reported in leaves fraction of carrot (Murugan et al., 2014). The difference in results is due to the use of difference in reporting units, nature of sample and cultivar in b other studies. Hydroxyl radical is the most reactive among all forms of dioxygen and initiative of cell damage (Duan et al., 2007). Scavenging of hydroxyl radical is an important activity due to vary high reactivity of OH radicals. Hydroxyl radicals have ability to react with macromolecules of living cells (Wang et al., 2008). The data regarding hydroxyl radical capacities of top whole of selected cultivars of D. carota are presented in Table 4.14. It showed that lowest hydroxyl radical capacity (26.88mg/100g) was found in DCY cultivar and that of highest (51.69mg/100g) in DCP cultivar. The findings related to hydroxyl radical capacity in top whole of selected cultivars of D. carota of present study are not comparable to previously published results (29.3 to 84%) in leaves of Bouhinia vahlii Wight and Arn. The impossibility of comparison to the results in literature is due to the difference in interpretation of evaluation methods and measuring units. From the findings of the present study regarding free radical scavenging capacities it may be concluded that top whole of selected cultivars of D. carota has significant level of antioxidants naturally. The antioxidant nature of these cultivars may be due to the presence of phenolic acids or other antioxidant compounds. Thus these top whole can be considered as new source of antioxidants and can be used in the development and production of valuable food additives for research organization and process industries.

4.3.3 Antioxidant activities of seeds extract D. carota 4.3.3.1 Total phenolics (TP) Extracted from carrot seed have protective effects against cardiovascular, hepatic, bacterial and fungal diseases due to the presence of phenolic and other beneficial compounds (da Silva Dias, 2014). The Data regarding total phenolics in carrot seed oil of selected cultivars of D.

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carota is presented in Table 4.15. It showed that lowest phenolics contents (6.13mg/100g) were found in DCY cultivar and that of highest (22.15mg/100g) in DCP cultivar. Statistical analysis revealed that total phenolic acid contents varied significantly (ρ < 0.05) among seed extracts of D. carota cultivars. The findings related to phenolic acid contents in seed extracts of D. carota cultivars is not comparable to previously published results (7.08 mg GAE/g) in carrot seeds collected from Algeria (Ksouri et al., 21015). The findings regarding the total phenolic acid contents of present study cannot be compared with cited literature due to difference in cultivar types, interpretation of results in different units and insufficient data on seed extracts of carrot. The phenolic acid contents in selected D. carota cultivars are comparable to other seed extracts reported in literature i.e. 0.119mg/mL in Agriophyllum pungens seeds (Birasuren et al., 2013) and 117.0 -177.4 mg GAE/L in pomegranate seed extracts (Gozlekc et a., 2011). The findings regarding the total pheonlic acid contents revealed the fact that seeds of selected D. carota have much potential against oxidative problems and can be used in valuated products and functional food.

4.3.3.2 Total antioxidant capacity (TAC) The data regarding total antioxidant capacity (TAC) of selected cultivars D. carota seeds are presented in Table 4.15. It showed that highest TAC (61.31mg/100g) was found in DCP and that of lowest (28.21mg/100g) in DCY cultivar. Statistical analysis revealed that total TAC varied significantly (ρ < 0.05) among seed extracts of D. carota cultivars. These findings regarding TAC were not comparable to previously published results (200-400 mg/kg) reported in carrot seed extracts (Rezaei-Moghadam et al., 2012). On the other hand both studies confirm the fact that carrot seeds extract have significant potential to increase the antioxidant status in living cells. Among the selected cultivars of the present study seed extract of DCP cultivar showed higher level of TAC as compare to other cultivars. In view the findings of the present study regarding TAC, it may be concluded that seed extract of selected cultivars have much potential to enhance the antioxidant capacity in animals or human beings.

4.3.3.3 Free radical scavenging capacities Living organisms defend against oxidative problems by antioxidant enzymes, antioxidant food constituents and antioxidant acts. Antioxidants in both forms, natural or synthetic are

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effective against these problems, reduced the rate of free radicals formations in living cells (Valko et al., 2006). The data regarding DPPH scavenging capacity of selected cultivars seed extracts of is presented in Table 4.15. It showed that highest DPPH scavenging capacity (37.61mg/100g) was found in DCP cultivar and that of lowest (12.35mg/100g) in DCY cultivar. Statistical analysis revealed that DPPH scavenging capacity varied significantly (ρ < 0.05) among seed extracts of D. carota cultivars. The presence of significant potential for DPPH scavenging capacity in seed extract of selected cultivars in the present study is comparable to the fact that seed extracts having antioxidants, can be used for curing lipid oxidation, maintaining nutritional quality and prolonging the shelf life (Ak & Gülçin, 2008; Ahmida, 2012). The data regarding superoxide radical scavenging capacity in seed extracts of selected D. carota cultivars is presented in Table 4.15. It showed that highest of superoxide radical scavenging capacity (38.16mg/100g) was found in DCP and that of lowest (13.35mg/100) in DCY cultivar. Statistical analysis revealed that superoxide radical scavenging capacity varied significantly (ρ < 0.05) among seed extracts of D. carota cultivars. The results regarding superoxide radical scavenging capacities showed that seeds extract of selected cultivars is a good source of antioxidants which can be used to retard the oxidation of fatty acids by scavenging the superoxide radicals (Zhishen et al., 1999). The data regarding the hydroxyl radical scavenging capacity of seed extract of selected D. carota cultivars is presented in Table 4.15. It showed that highest hydroxyl radical scavenging capacity (37.70mg/100g) was found in DCP cultivar and that of lowest (12.89mg/100g) in DCY cultivar. Statistical analysis revealed that hydroxyl radical scavenging capacity varied significantly (ρ < 0.05) among seed extracts of D. carota cultivars. Body metabolism also results in the production of hydroxyl radicals as byproducts, dangerous to biological molecules results in degenerative diseases (Valentao et al., 2002). The findings regarding the hydroxyl radical scavenging capacity in present study prove that seed extracts of selected cultivars have significant potential to cure or to prevent diseases caused by hydroxyl radicals. In view of findings related to antioxidant activities of seeds extract D. carota and correlative effects on living cells in literature, it may be concluded that seed extracts of selected cultivars have significant potential to cure the diseases, resulted from oxidation process or metabolism in living cells. Seeds of cultivars under study may also be used in process industries for extraction and purification of antioxidants, for the production of anticancer medicines and functional foods.

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Table 4.14 Antioxidant activities of selected cultivars of D. carota top whole Total Antioxidant DPPH scavenging Superoxide radical Hydroxyl radical Types Total phenloic capacity capacity scavenging capacity scavenging capacity D. carota acid content (mg/100g)* (%)* (mg/100g)* (mg/100g )* cultivars (mg/g)*

DCW 7.20 ±0.13b 42.11±0.81b 25.56±0.75b 26.26±0.82b 27.89±0.96b

DCY 6.13 ±0.24a 40.41±0.87a 24.55±0.82a 25.23±0.93a 26.88±0.84a

DCP 22.15 ±1.81g 73.51±2.82g 49.81±1.13g 50.06±1.52g 51.69±1.98g

T29 11.71±0.34e 51.97±1.96e 29.73±0.79e 30.51±0.92e 32.14±0.91e

DCR 8.16±0.31c 44.45±1.25d 26.89±0.91d 27.59±0.79d 29.22±0.89d

DC3 12.79±0.92f 54.38±1.67f 35.36±1.02f 36.06±1.13f 37.69±1.04f

DC90 9.41±0.27d 44.2±0.84c 28.33±0.89c 29.03±0.91c 30.66±0.96c

*The findings are described as mean ± standard-deviation. The super-script-lettering in editorial illustrate noteworthy-difference (ρ < 0.05).

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Table 4.15 Anti-oxidant-activities of seed-extorts of diverse D. carota (carot) cultivars phenloic acid Antioxidant scavenging activity scavenging activity scavenging activity Extracts of D. content (Total) activity (Total) (DPPH) (Superoxide radical) (Hydroxyl radical) carotacultivars (mg/g)* (mg/100g)* (%)* (mg/100g)* (mg/100g )*

9.70 ±0.13b 29.91±0.41b 13.36±0.45b 14.36±0.61b 13.90±0.44b DCW

7.11 ±0.21a 28.21±0.38a 12.35±0.52a 13.35±0.63a 12.89±0.41a DCY

42.24 ±1.71g 61.31±2.42g 37.61±1.66g 38.16±2.32g 37.70±1.68g DCP

13.77±0.16e 39.77±0.65e 17.61±0.59e 18.61±0.62e 18.15±0.51e T29

10.06±0.11c 32.25±0.55d 14.69±0.51d 15.69±0.60d 15.23±0.79d DCR

DC3 19.49±1.10f 42.18±1.72f 23.16±1.52f 24.16±1.63f 23.70±1.54f 15.41±0.28d 32.00±0.54c 16.13±0.53c 17.13±0.61c 16.67±0.56c DC90

*The values are presented in mean ± standard-deviation. The superscript-letters in the columns show significant-difference (ρ < 0.05).

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Part IV 4.4 Physical analysis of carrot juice Juice of fruits and vegetables provides ways to access fiber locked and fiber incorporated enzymes, beneficial to enhance digestion process (Murray, 2013). Fresh juices are also loaded with a lot of minerals, phytonutrients and vitamins (Calbom, 2011). Due to the presence of these valuable attributes juices of fruits and vegetables are effective against premature aging and enhance immune system and show preventive effect against many types of diseases like cardiovascular and stomach problems (Cencic & Chingwaru, 2010). Physical analysis of juices is valuable tool to explore its physical properties. The data regarding the physical analysis of carrot juice is presented in Table 4.16. pH measurement is important in medicine, chemistry, biology, agriculture, food science etc. It is the imbalance of pH (acidic or basic) that allows unhealthy microorganisms to damage body tissues and ultimately affect the immune system (Elgert, 2009). The data regarding pH of juice of selected cultivars of D. carota showed that pH varied from 4.73 for DC3 to 5.53 for DCP cultivar.Nirmala et al. (2011) reported pH values of some common Juices: pineapple (3.3-3.7), orange (3.26-3.75), mango (4.20-4.6) and sugar can (4.20-4.6). Reported pH values for common fruits juices is lower than that of found in present study, but on the other hands, the recommended value for drinking water is from 6.5-8.5 for surface water and 6-8.5 for ground water (Kazi, et al., 2009). The values obtained for pH in the present study are near to recommended values for drinking water. From the findings of the present study, it may be concluded that carrot juice (DCP cultivar) has body balancing properties like drinking water and has ability to maintain the pH of body fluids(Bossingham et al., 2005). The total soluble and total solids are the quality parameter for certain types of juices (Stintzing et al., 2003). The findings related to total soluble and total solids in juices of selected cultivars of D. carota are presented in Table 4.16. The total soluble in juices of D. carota cultivars varied from 7.35% for DCR to 9.10% for DCP cultivar and the total solids varied from 7.88% for DCR to 9.56% for DCP cultivar. The findings of present study related to total soluble and total solids are not comparable to the previously published results: total solids (6.0 %) and total soluble (5.6%) in water blended fresh carrot juice (Waghray et al., 2012), but another study shows total solids (8.50%) in fresh carrot juice, which is comparable to the results of present study (Rashidi & Gholami, 2011). The difference in results is due to preparation method of juice in both studies. From the findings of present study related to total

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soluble and total solids it may be concluded that total soluble and solids can be used for the determination of carrot juice purity. Acidity has a leading role not only in regulating the biological processes but also chemical reactions (Xu et al., 2006). The findings regarding the acidity in juices of selected cultivars of D. carota are presented in Table 4.16. Acidity in juices of selected cultivars ranged from 0.08mg/100mL for DCP to 0.13mg/100mL for DC3 cultivar. The findings regarding the acidity of selected cultivars are comparable to the acidity result (0.10mg/100mL) of red carrot juice (Leahu et al., 2013). Acidities of lemon (1.44g/ounce) and lime (1.3844g/ounce) juices are due to the presence of citrus acid (Penniston et al., 2008), which can cause acid reflux (Souza et al., 2009). In-comparison carrot juice of selected cultivars is less acidic than citrus juices. It may be conclude from the findings of present study regarding acidity, juices of D. carota cultivars can be used for avoiding or curing the acid reflux diseases. Refractive index and brix are used to identify substances by measuring their purities and concentrations in aqueous or solution forms (Brar & Verma, 2011). There is positive relationship between refractive index and brix (Baldini et al., 2004). The data regarding the refractive index and brix of juices of selected cultivars of D. carota is presented in Table 4.16. Refractive index of selected cultivars ranged from 1.3418 for DCR to 1.3462 for DCP cultivar and the brix values ranged from 9.92% for DCR to 11.52% for DCP cultivar. The relationship between refractive index and brix in the present study is in-agreement as described by Baldini et al. (2004). The findings regarding the refractive index are comparable to the previously results (1.3516) for carrot juice (Leahu et al., 2013). There is also a positive correlation between brix and carbohydrate concentration (Audilakshmi et al., 2010). From the findings of present study regarding refractive index and brix, it may be concluded that juices of selected cultivars have considerable amount of carbohydrates, sufficient for energy requirement for human body. The findings related to juice of selected cultivars of D. carota showed that carrot juice has significant potential, not only against cardiovascular and gastrointestinal diseases like citrus juices without causing acid reflux but also may be used as energy drink due to high carbohydrates contents. Taking into consideration the above findings regarding selected cultivars of D. carota, a study was arranged on blood of industrial workers with advancing age.

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Table 4.16 Physical properties o of D. carota juice collected from selected cultivars of D. carota roots

D. carota Cultivars

Parameter T29 DCR DC3 DC90 DCY DCP DCW

pH 5.12±0.07f 4.92±0.06b 4.73±0.05a 5.03±0.06d 5.10±0.05e 5.53±0.08g 4.95±0.05c

Total soluble % 8.15±0.13c 7.35±0.34a 8.13±0.35f 8.05±0.33e 8.11±0.18d 9.10±0.15g 7.87±0.16b

Total solids% 8.56±0.16e 7.88±0.19a 8.82±0.20f 8.46±0.14d 8.41±0.13c 9.56±0.17g 8.08±0.17b

Acidity 0.11±0.02b 0.13±0.03e 0.13±0.02d 0.11±0.04d 0.12±0.02c 0.08±0.01a 0.11±0.04d mg/100mL

Refractive index 1.3433± 1.3418± 1.3431± 1.3424± 1.3429± 1.3462± 1.3421± (25) 0.0002f 0.0003a 0.0002e 0.0002c 0.0003d 0.0003g 0.0002b

Brix (%) 10.51±0.13e 9.92±0.14a 10.83±0.15f 10.43±0.12c 10.46±0.15d 11.52±0.12g 10.11±0.12b

*The values are presented in mean ± standard-deviation. The superscript-letters in the columns show significant-difference (ρ < 0.05).

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4.5 Effect of carrot juice on human blood D. carota have high value of fiber, vitamin C, vitamin E, phenolics and carotenoids as compare to other vegetables (Alasalvar et al., 2001). It has been approved by the previous studies that foods containing phenolic acids decrease the risk of cardiovascular diseases (Schroeter et al., 2006).Table 4.17-4.20 presented the pre-study and post-study physical parameters, blood chemistry, lipid profile, and antioxidant status of volunteers of various age groups. Post study data of all age groups showed that advancing age increased the body weight and affected the BMI significantly. Obesity and high BMI increases the risk of cardiovascular diseases and diabetes (Lin et al., 2008). Drinking D. carota juice for thirty days decreased the obesity level insignificantly (ρ ≥ 0.05). These findings are not comparable to the results reported by Potter et al. (2011), in which they reported that no change was observed in body weight and BMI after drinking carrot juice for 90 days. This may be due to elevated cholesterol, life style and gender difference. All the treatment groups have high blood pressure due to intense environment of industry and effect was increased with advancing age. Drinking 250mL D. carota juice lower diastolic blood pressure significantly (ρ < 0.05) whiles an insignificant change was observed in diastolic pressure. Up to 4.64 % and 9.64% systolic blood pressure was decreased in treatment group 1 and 4 individuals respectively, while potter et al. (2011) reported 5.0 % reduction in systolic blood pressure after drinking carrot juice for 90 days. This may be due to difference in carrot variety, individual age and duration of the study. Advancing age in industrial environment changed the blood plasma chemistry, a major cardiovascular risk factor. With advancing age sodium level decreased (Table 4.17- 4.20). Depletion in sodium is associated with cardiovascular problems (Shannon et al., 1986). Drinking 250mL D. carota juice increased sodium level (~3.26%) in all age groups. Potassium level increased with advancing age in industrial workers which is in agreement with Doorenbos and Vermeij (2003), they reported that potassium level increased in blood with advancing age. The increase in potassium level in blood cause excretion problems like kidney failure (Doorenbos & Vermeij, 2003). In the presented study it has been found that drinking D. carota juice maintained the potassium level in blood in all age groups. This finding was in agreement with Costa et al. (2004), reported that carrot has much potential for increasing potassium level in blood. Calcium Concentration decreased with advancing age (Table 4.17-4.20).

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The calcium is beneficial for bones, teeth, heartbeat, blood pH and blood pressure (Sharma et al., 2011). Drinking carrot juice for thirty days increased the calcium level in blood, by which blood pressure and other cardiovascular risk factors could be controlled. These findings are in agreement with Sharma et al. (2011); they reported that wheat juice reduced the cardiac problems by maintaining calcium level in blood. Albumin concentration decreased with advancing age (Table 4.17-4.20). Albumin binds water, K, Na and calcium ions hence it normalize the blood osmotic pressure (Farrugia, 2010). Drinking D. carota juice increased the albumin in industrial workers to regulate blood functions. But the Potter et al. (2011) reported so as to carrot fluid did not affect the albumin in woman. With advancing age blood glucose was increased (Table 4.11 to 4.14). Although sugars are present in carrots but it showed minor increased (ρ > 0.05) in glucose level in higher age individuals of group4. Potter et al. (2011) reported decreased in blood glucose after drinking carrot juice. The minor increase in blood glucose may be due to the presence of herbal insulin in D. carota (Baranska et al., 2005). The difference in findings may be due to eating habits of the personals. Red blood cells (RBC), white blood cells (WBC) and plate level decreased significantly (ρ < 0.05) which resulted in the lowering of blood volume, hemoglobin and hematocrite in industrial workers with advancing age (Table 4.17-4.20). These results are in agreement with Silver et al. (1999); they reported that blood volume decreased with advancing age due to lesser production of blood cells by bone marrow. The gradual decrease in hemoglobin results in anemia (Allen, 2000). Drinking D. carota juice increased the blood cells, hemoglobin and hematocrite in industrial workers with advancing age. This was in agreement with the findings that carrot, apple and tomato juice increased the blood cells and volume (Press, 2014). Blood lipid profile including triglyceride, cholesterol, high density lipoprotein (HDL), very low density lipoprotein (VLDL) and low density lipoprotein (LDL) increased significantly (ρ < 0.05) in industrial workers with advancing age presented in Table 4.17 to 4.20. Triglycerides are the main component of fat inhuman, animals and vegetables, enabling the bidirectional movement of fat and glucose from liver to body organs (Nelson & Cox 2000). High concentration of triglycerides is the cause of diabetes and kidney diseases (King et al., 2015). Drinking D. carota juice maintained the level (ρ < 0.05) of triglycerides in industrial workers with advancing age. The decrease in triglycerides in blood after drinking D. carota juice was not comparable to the findings reported by Vafa et al. (2011); they reported insignificant increase was observed in triglycerides after eating apple for eight

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weeks. On the other hands Ataie-Jafari et al. (2008) found that triglycerides decreased after drinking cherry juice for six weeks. The difference in results is due to the difference in juice origin or may be due to the eating and health habits of the subjects under study. Cholesterol level increased significantly (ρ < 0.05) in industrial workers with advancing age. The increase in cholesterol level in blood results in the heart diseases (Laskowski, 2012). Drinking carrot juice in advancing age decreased or maintained the level of cholesterol in blood (Table 4.17-4.20). The decreased in cholesterol level was comparable to the findings reported by Esmaillzadeh et al. (2004); they reported that cholesterol level decreased significantly (ρ < 0.05) after drinking 40g/day of pomegranate juice in diabetic patients. The LDL and VLDL transport the fat from liver to body cells. The high level of LDL and VLDL in blood increases the risk of atherosclerosis which causes heart attack and stroke (Feuerstein, 2016). LDL and VLDL increased significantly (ρ < 0.05) in industrial workers with advancing age presented in Table 4.11 to 4.14. Drinking D. carota juice decreased or maintained the LDL and VLDL level in industrial workers with advancing age significantly (ρ < 0.05). The decrease in LDL and VLDL with carrot juice is not comparable to the findings of potter et al. (2011); they reported carrot juice didn’t affect the blood lipid profile of the individual having disturbed lipid profile. The difference in results is due to the difference in juice origin or may be due to the eating and health habits of the subjects under study. Plasma antioxidant status decreased in industrial workers with advancing age,the decrease in antioxidant status was comparable to findings, reported by Limberaki et al. (2012) that antioxidant status decreased from young to old age. Drinking D. carota juice increased antioxidant capacity of blood; it may be due the presence of considerable amount of phenolic compounds and other anti-oxidants (Kumarasamy et al., 2005).These findings are comparable to antioxidant capacity reported by Tonin et al. (2015); they found that antioxidant capacity of blood increased after dinking natural juices of different fruits and vegetables. The young individuals of group 1 had more increased in antioxidant capacity than old age individuals of group 4. But Limberaki et al. (2012) reported the reverse effect after taking antioxidant diet. This may be due to diet difference and routine eating habits. Malondialdehyde production was increased in industrial workers with advancing age (Table 4.17- 4.20). This was in agreement with Sayyah (2011); he reported that malondialdehyde production was increased in asthmatic patients with advancing age. Drinking carrot juice decreased the malondialdehyde production significantly (ρ < 0.05) in young (37.12%) as well

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as in old (26.05%) age groups (Table 1-4). Decreasing trend of malondialdehyde is comparable to potter et al. (2011), while they reported >50% reduction in malondialdehyde production after drinking carrot juice for 90 days. The difference in findings may be due the duration of study and carrot variety used in both studies. The lesser increase in antioxidant capacity and lesser decrease in malondialdehyde production in the present study as compare to other studies may be due to the difference in environments and eating habits.

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Table 4.17 Physical parameters, blood chemistry, lipid profile, and antioxidant status of volunteers of treatment group No 1 (20 to 30 years) At the time of % Increase/ Variables After 30 days beginning decrease Body weight (Kg) 65.00 ± 5.66 63.75 ± 5.30 -2.28 Height (cm) 170 ± 14 170 ± 14 0.00 BMI 21.85 ± 0.78 22.10 ± 1.84 5.79 Blood Pressure Systolic (mm Hg) 123.7 ± 2.36 119.5 ± 0.71 -4.64 Diastolic (mm Hg) 78.5 ± 2.1 79.0 ± 1.41 -0.24 Blood Chemistry

Na’ (mmol’/L)’ 131.5 ± 0.71 135.0 ± 1.41 3.18

K’ (mmol’/L) ’4.26 ± 0.04 ’4.21 ± 0.03 -1.40

C’a (mg’/dL ) ’9.65 ± 0.06 ’9.78 ± 0.07 1.44

Albu ’ min’ (g/’dL) ’4.55 ± 0.07 ’4.82 ± 0.08 6.06

Gluc’ose’’ (mg/’ dL) ’89.5 ± 2.12 ’92.5 ± 2.71 3.92 Red blood cells (×106/µL) 49.5 ± 0.71 52.5 ± 0.34 5.24 White blood cells (/µL) 5250 ± 247 5370 ± 311 3.35 Hemoglobin (g/dL) 13.65 ± 0.21 14.50 ± 0.07 5.12 Hematocrit (%) 40.95 ± 0.64 42.15 ± 0.71 3.05 Platelet (×103/µL) 388.5 ± 14.8 420.5 ± 13.2 7.54 Lipid Profile Triglyceride (mg/dL) 113.4±9.4 115.2±9.2 1.30 Cholesterol (mg/dL) 225.5±27.3 224.3±25.9 -1.03 HDL (mg/dL) 46.9±7.3 45.6±7.1 -2.77 VLDL (mg/dL) 29.9±8.5 30.2±8.1 -0.26 LDL (mg/dL) 128.4±13.5 127.4±13.0 -1.06 Antioxidant status Antioxidant capacity (mM) 0.88 ± 0.05 1.12 ± 0.07 27.96 Malondialdehyde (μM) 44.0 ± 5.33 29.1 ± 1.92 -37.12 All *Means ± SD are significantly-different (ρ < 0.05)

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Table 4.18 Physical parameters, blood chemistry, lipid profile, and antioxidant status of volunteers of treatment group No 2 (31 to 40 years) At the time of % Increase/ Variables After 30 days beginning Decrease Body weight (Kg) 69.50 ±10.61 68.75 ± 10.25 -1.39 Height (cm) 167 ± 2.83 167 ± 2.83 0.00 BMI 24.9 ± 2.97 24.6 ± 2.83 -1.58 Blood Pressure Systolic (mm Hg) 126 ± 2.41 119.5 ± 0.71 -6.39 Diastolic (mm Hg) 84 ± 2.83 79.0 ± 1.41 -7.39 Blood Chemistry Na (mmol/L) 128.0 ± 3.54 129.0 ± 3.58 0.79

K’ (mmol’/L) ’4.49 ± 0.16 ’4.30 ± 0.07 -6.02

C’a (mg’/dL ) ’9.31 ± 0.71 ’9.80 ± 0.74 5.19

Alb’umin’ (g/’dL) ’4.33 ± 0.07 ’4.37 ± 0.07 0.91

Glu ’cose’’ (mg/’ dL) ’132 ± 5.1 ’137 ± 4.2 2.99 Red blood cells (×106/µL) 46.5 ± 0.71 490 ± 0.14 4.09 White blood cells (/µL) 5075 ± 212 5450 ± 118 5.31 Hemoglobin (g/dL) 13.05 ± 1.20 13.70 ± 0.85 2.11 Hematocrit (%) 39.15 ± 3.61 41.10 ± 2.55 2.08 Platelet (×103/µL) 378.0±8.48 405.5 ± 7.78 6.93 Lipid Profile Triglyceride (mg/dL) 135.4±28.5 134±25.8 -2.50 Cholesterol (mg/dL) 252.6±25.4 248±21.2 -3.17 HDL (mg/dL) 48.4±7.8 47.1±6.9 -3.91 VLDL (mg/dL) 30.5±7.1 29.1±7.0 -3.99 LDL (mg/dL) 139.5±12.2 138.3±11.4 -1.32 Antioxidant status Antioxidant capacity (mM) 0.84 ± 0.04 0.99 ± 0.06 19.32 Malondialdehyde (μM) 46.0 ± 7.63 28.3 ± 6.52 -35.07 All *Means ± SD are significantly-different (ρ < 0.05)

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Table4.19 Physical parameters, blood chemistry, lipid profile, and antioxidant status of volunteers of treatment group No 3 (41 to 50 years) At the time of % Increase/ Variables After 30 days beginning decrease Body weight (Kg) 79.33±9.07 78.33 ± 8.58 -1.69 Height (cm) 157.67±4.93 157.67± 4.93 0.00 BMI 31.87±2.56 31.47 ± 2.40 -1.63 Blood Pressure Systolic (mm Hg) 132.33±4.16 124.69 ± 2.52 -6.80 Diastolic (mm Hg) 86.0±2.00 81.0 ± 2.65 -4.96 Blood Chemistry Na (mmol/L) 122.67±2.52 124.09 ± 2.0 0.72

K’ (mmol’/L) ’4.80±0.07 ’4.47 ± 0.06 -6.98

C’a (mg’/dL ) ’8.83±0.15 ’9.13 ± 0.21 4.01

Al ’ bumin’ (g/’dL) ’4.28 ± 0.09 ’4.67 ± 0.11 9.38

Gluc’ose’’ (mg/’ dL) ’137.3±19.10 ’144.3 ± 20.2 5.18 Red blood cells (×106/µL) 44.4± 1.53 49.0 ± 1.73 3.89 White blood cells (/µL) 4920.3±79.4 5076.0 ± 88.9 2.79 Hemoglobin (g/dL) 12.97±0.89 13.57 ± 0.90 4.40 Hematocrit (%) 38.9±2.69 40.7 ± 2.69 4.33 Platelet (×103/µL) 254.33±3.51 258.33 ± 15.37 6.15 Lipid Profile Triglyceride (mg/dL) 163.7±19.5 160.2±17.4 -3.06 Cholesterol (mg/dL) 254.6±17.9 248.6±13.2 -3.93 HDL (mg/dL) 50.4±8.5 48.3±7,9 -4.58 VLDL (mg/dL) 33.9±6.6 31.6±7.1 -4.44 LDL (mg/dL) 144.5±10.4 140.2±9.6 -3.29 Antioxidant status Antioxidant capacity (mM) 0.71 ± 0.07 0.89 ± 0.06 21.79 Malondialdehyde (μM) 50.20 ± 6.83 36.33 ± 3.32 -30.48 All *Means ± SD are significantly-different (ρ < 0.05)

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Table4.20 Physical parameters, blood chemistry, lipid profile, and antioxidant status of volunteers of treatment group No 4 (51 to 60 years) At the time of % Increase/ Variables After 30 days beginning decrease Body weight (Kg) 80.00±5.29 78.47 ± 3.14 -4.31 Height (cm) 166.0±5.29 166.0 ± 5.29 0.00 BMI 29.03±0.71 28.47 ± 0.55 2.42 Blood Pressure Systolic (mm Hg) 141.67±3.51 129.67 ± 1.52 -9.64 Diastolic (mm Hg) 90.0±2.0 87.67 ± 2.1 -2.42 Blood parameters Na (mmol/L) 120.67±1.53 123.67 ± 2.51 3.26

K’ (mmol’/L) ’5.22±0.03 4.78 ± 0.04 -8.19

C’a (mg’/dL ) ’8.71±0.15 9.0 ± 0.18 1.77

Alb’umin’ (g/’dL) ’3.96 ± 0.11 4.05 ± 0.14 2.95

Glu ’cose’’ (mg/’ dL) ’152.3±31.6 154.7 ± 32.3 1.69 Red blood cells (×106/µL) 41.5± 0.58 44.67 ± 0.42 7.15 White blood cells (/µL) 4850±85.4 4880.0 ± 95.9 5.08 Hemoglobin (g/dL) 12.6±0.36 12.97 ± 0.45 3.55 Hematocrit (%) 37.8±1.10 38.9 ± 1.76 4.52 Platelet (×103/µL) 228.33±2.31 249.34±2.46 9.17 Lipid Profile Triglyceride (mg/dL) 210.6±34.5 201±32.1 -4.90 Cholesterol (mg/dL) 295.4±29.9 281.3±28.1 -4.89 HDL (mg/dL) 53.4±8.5 51.2±7.6 -5.01 VLDL (mg/dL) 49.8±7.4 47.5±6.8 -5.07 LDL (mg/dL) 162.5±15.2 156.6±12.3 -4.95 Antioxidant status Antioxidant capacity (mM) 0.66 ± 0.02 0.86 ± 0.02 29.41 Malondialdehyde (μM) 51.90 ± 5.83 41.37 ± 1.32 -26.05 All *Means ± SD are significantly-different (ρ < 0.05)

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Chapter 5 SUMMARY The research work presented in dissertationtitled “Physico-chemical characterization and biological activity of Carrot (Daucus carota) cultivars indigenous to Pakistan’ was performed in the laboratories of Department of Applied Chemistry and Biochemistry, Government College University, Faisalabad, Prime Care Clinical Computerized Lab., Faisalabad, Pakistan and Quality Operation Laboratory (QOL), University of Veterinary and Animal Sciences, Lahore. A total of seven (7) culativars of carrot were obtained from the fields of Ayub- Agriculture Research-Institute (AARI), Faisalabad, Pakistan. The present desertation consists of four main sections: introduction, review of literature, methodology and results discussion. First section (chapter 1) describes the research subject, importance, approach, contribution to the prvious researches, logical idea behind this research and objectives of the research. Second section (chapter 2) demonstrates the theoretical framework of the project, identified the case studies, models and define the area of research. This section also described the methodology used in previos works and demonstrated the gaps in the previous works and how the present topic indens to fill the gaps. Third section (chapter 3) demonstrates the sample collection, pretreatments and preparation for certain analysis. This section also describes the use of several types of analytical mehods or tools including high performance liquid chromatography (HPLC) coupled to different detectors including refractive index (RID) and diode array detector for the dectection and quantification of phenolics and sugar compounds. Some of analytical methods were performed as such and some were performed after little modifications. The fouth section (chapter 4) has forther been divided into four parts to demonstrate the results and discussion. Part I demonstrates the comparative proximate composition of root, seed and top whole among new cultivars and previously cited works about D. carota. This part also explaines the physico-chemical properties of D. carota seed oil including acidity, peroxide value, saponification number, unsaponification number, specific garavity and refreactive index. Part II consists of findings about idnentification and quantification of individual sugars and phenolic compounds by using chrpmatographic techniques. Part III demonstrates the quantification of beta-carotenes and total phenolic acids and antioxidant

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activity (total antioxidant activity, DPPH scavenging capacity, super radical scavenging capacity and hydroxyl radical scavenging capacity) of root and top whole of selected cultivars of D. carota using spectrophtometric technique. Effects of carrot juice on human obesity and blood are demonstrated in part IV under the headings of body weight, hight, body mass index (BMI) blood-pressure, blood-chemistry, lipid profile and blood-antioxidant status. All the investigated cultivars of D. carota showed good proximate profile viz. moisture (86.6 to 92.89%), proteins (0.56 to 1.68%), crude fibers (1.55 to 3.28%), ash (0.40 to 1.20%), carbohydrates (6.44 to 8.13%), fats (0.27 to 0.46%) and Calorific energy (26.38 to 38.42 kcal/100g). The mineral contents determined by atomic absorption spectrophotometer (AAS) viz. Co (0.19 to 0.48µg/g), Cu (1.20 to1.99µg/g), Fe (4.01 to 5.90µg/g), Sr (2.94 to 4.17µg/g) and Zn (2.0 to 3.15µg/g). Spectrophotometric analysis presented the appreciable level of β-carotene (6.12 - 14.87 mg/100g) proving the medicated properties of newly invented cultivars of D. carota, however DCP showed excellent potential for functional and dietary supplementation. These newly developed cultivars may be recommended that consumers should use adequate amount of D. carota to fulfill the essential dietary requirements to cope the existing challenges of malnutrition. Furthermore, β-carotene, Zn and crude fiber owing their health benefits furnish the functional food status to D. carota. Characterization, quantification and identification of phenolic-compounds secluded from selected D. carota cultivars were completedearlier to antioxidant-activity i.e. analysis on RP-HPLC revealed that hydroxycinnamic acids and its derivative-forms were major

phenolic compounds present in the extract ofDaucus carota whereas 5 ‘-‘ caffeolquinic acid

was a major ‘ hydroxycinnamic-acid (varied from 30.26 - 65.39 mg/100g). The selected cultivars showed high variation in the contents of total phenolics (30.26 - 65.39 mg/100g) and total ascorbic acid (41.12 - 58.36 mg/100g). The hydroxycinnamic-acids and its derivative-forms were found in significant concentrations. Concentration of phenolic compounds differed from cultivar to cultivar, Phenolic-acid contents and anti-oxidant activity correlated well i.e. an increase in phenolic-acid concentration results in the increase of antioxidant activity and vice versa.. DCP cultivar showed high total antioxidant capacity (77.69mg/100g), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity (52.36mg/100g), superoxide-radical scavenging-capacity (53.69mg/100g and hydroxyl-

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radical scavenging-capacity (51.91mg/100g). Therefore, it can be concluded from the current study that contents of phenolic-acids are the dependent parameters which revealed the antioxidant properties of certain plant materials. DCP cultivar might be painstaking for considerable utilization in the doling-out–production owing to the existence of high -strength of phenolic-acids and anti-oxidant activitiesin-comparison to remaining-cultivars of current study. The developed method using HPLC with RID proved to be a swift and proficient technique for quantification and detection of individual sugars. The concentration (p < 0.05) of total saccharides in requisites of maltodextrin , fructose, maltotriose, maltose, and glucose in selected verties (DCW, DCY, DCP, T29, DCR, DC3 and DC90) were 04.511, 05.165, 06.311, 05.281, 04.912, 05.099 and 04.448g/100g respectively. Preparation for sample was easy and purified water was used as mobile phase, produced nonhazardous scum. Limits of detection and quantification are consistent which were in the range of 48.13 to 59.45 mg/L and 49.36 to 178.23 mg/L respectively. Recovery of sugars was > 90%. All validation steps proved that the developed method is efficient, reliable, economical and environmental friendly. DCP cultivar has premier sugar intensity in contrast to further cultivars. Consequently, DCP cultivar might be utilized in consign of other D. carota (cultivars) varieties, to get benefit of its nutraceutical attributes. In industrial workers, oxidative stress with advancing age changed the blood composition and obsety. Drinking D. carota juice decreased the body weight and body mass index (BMI) insignificantly (ρ ≥ 0.05). It increased blood cells, platelets, hemoglobin, hematorite, sodium, calcium, albumin and glucose levels significantly (ρ < 0.05) but potassium level decreased insignificantly (ρ ≥ 0.05) with advancing age. D. carota juice decreased systolic pressure, but minor effect was observed in diastolic pressure. Drinking D. carota juice significantly decreased(ρ < 0.05) triglyceride, cholesterol, HDL, LDL and VLDL among treatment groups. Total antioxidant-capacity blood-plasma increased significantly but production of malondialdehyde was decreased. The D. carota juice has much potential owing to functional compounds which had remedial action against irregularities in blood and body functions.

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Conclusion This desertation systematically scrutinized physico-chemical composition and biological activity of newely invented cultivars of D. carota. These cultivars showed remarkable amount of functional attributes in terms of protein, carbohydrates, oil, minerals, crude fiber, phenolic and carotenoid compounds. D. carotacultivars showed variations in the concentrations of Phenolic-acids, accountable to dissimilarities in antioxidant-properties proving direct correlation between phenolic-acid contents and antioxidant-activities. Novel cultivars of D. carota found to be considerable sources of sugars including maltodextrin, maltotriose, maltose, fructose and glucose, beneficial for regulating the proper body functions in human beings. In carrots these sugars fuction to protect membranes, proteins and the electron transport chain of photosynthesis under freezing and heat stresses.These cultivars showed considerable differences in functional attributes regarding difference in genetic background with inherent composition. These findings provide useful guidelines for breeding, cultivation and commercial utilization of carrot cultivars with special intentions interms of improving health promoting and sensory attributes.

Future prospects It is evident from the evaluated data that current study presents useful informations about proximates, carotenoids, minerals, phenolics, sugars compounds and biological activity of novel cultivars of D. carota. The deployment of indigenously cultivated carrot cultivars as potent source of antioxidants, vitamins, minerals and secondry metabolites ought to be encurged. Nevertheless, future studies under in-vivo conditions with variable amounts are suggested for futher elaboration as antioxidant, anti-microbial and anti-fungal actions to get persuasive remedial applications. The isolation of individual carotenoids, phenolics and sugar compounds at industrial or commercial is further suggested for the preparation of functional foods and nutraceutical applications. Developed method for sugars determination is applicable to quantify all types of sugars in D. carota. DCP cultivar contained higher concentration of sugars, it may be recommended in process industries for the extraction of dietary sugars of vegetal origin. Effective evaluation of individual bioactive compounds using different food substartes under local processing and evvironmental conditions is also recommended. It is recommended that production and concentration of these bioactive

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compounds in these cultivars should be increased by applying different types of agronomical approaches i.e. genetic and environmental approaches. Moreover, the protective cultivation of these cultivars at commercial scales is also suggested to get benefits of these cultivars throughout the year.

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