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Registration No: 2012-GCUF-01997
ABSTRACT
Micronutrient deficiencies have proven major contributors in the onset of different health related discrepancies and a woman throughout the life cycle is the most effective segment. Amongst various deficiencies, improper iron balance caused the onset of anemia, which hinders the optimum growth and development. The school going adolescence girls are often found anemic owing to changing dietary pattern, monotonous diet consumption and poor nutrition education. Enhance consumption of different fruits and vegetables are attained paramount position as a component of natural therapies for the anemia management. Considering the importance of fruits and vegetables, the current project is designed to evaluate the effectiveness of pomegranate, beet root and carrot for the management of anemia in school going adolescence girls. For the convenience, the study was divided into two experiments, in first experiment the development and physicochemical characterization of juice were done, whereas in proceeding experiment the bioefficiency trial was planned by involving the anemic school going girls to evaluate the effectiveness of developed intervention against this menace. In the first experiment, the beet root, carrot and pomegranate juice were prepared and evaluate physicochemical test like pH, acidity, and TSS and antioxidant profile. In the second experiment, 45 girls were recruited from the govt. Girls high school 197/R.B., Faisalabad. The selection is based upon specified inclusion and exclusion criteria according to WHO guidelines for such studies. The girls were divided into 9 groups (05 in each) and given the study treatments. The nine kinds of treatments including control will be prepared by utilizing all the juices alone as well as their combinations for a period of one month. The Consent Performa, the food frequency questionnaire Performa, Nutrition assessment like Anthropometric measurement, menstrual history, Clinical sign and symptoms , Anemia checklist and complete blood count CBC, Lipid profile, transferrin, ferratin, Transferin iron binding capacity TIBC was carried out at the initiation of trial (0th day). The all the biochemical analysis alongside the physical signs were conducted after 15th day and the 30th day. The obtained data was subjected to suitable statistical analysis.

Chapter # 1

INTRODUCTION
Micronutrient deficiencies are known as hidden hunger have been recognized as one of the major nutritional challenges being faced by the millions of people. It has been estimated that such nutritional imbalance affects about 1/3rd of the global population resulting in hampered growth and productivity (Siyame et al., 2013). Further into this situation, South Asian populations have the major share for poor growth, lowered cognitive performance and decreased immunity (Alexandre et al., 2014). It has been noticed that inadequate dietary selections, lack of awareness and poor socioeconomic status are the key reasons behind this menace.
Iron deficiency anemia is reported to affect nearly 2 billion people worldwide. Further, developing countries are challenged with some unusually high prevalence of anemia 43%. Furthermore, anemia is accounted for more than 115000 maternal deaths globally per annum (McLean et al., 2009; Sanghvi et al., 2010). Its deficiency has majorly been reported in regions with lower socioeconomic status like South Asia and Africa (Pasrichaet al., 2013). Infants and women (pregnant & lactating) are among the most vulnerable segment threatened by the onset of iron deficiency anemia (Lartey, 2008).
Pakistan is no exception to above and consistently facing the escalating incidence of micronutrient deficiencies. It is noteworthy to mention that food security crisis, low socioeconomic status and lack of adequate nutrition awareness further aggravate this situation (Baig-Ansari et al., 2008). To a cautious estimate, in Pakistan more than 60% preschoolers are confronted with iron deficiency. Likewise, around 35-40% women are iron deficient; of them 90% of pregnant women are threatened with low iron reserves thereby risking fetus survival and proper neonatal development. Although the situation in Pakistan is better as compared to other South Asian neighbors, but still there is a dire need to mitigate the prevalence of hidden hunger (WHO, 2012). Iron is among one of the vital micronutrients required for proper physiological functioning, mental development and provision of oxygen to the blood cells (Wang and Pantopoulos, 2011). Besides, it serves an integral component of many proteins and enzymes that ensure optimum physiological condition. In humans, iron is essential for cell growth and differentiation (Dwyer et al., 2003).

Iron deficiency is the most common cause of anemia. There are different types of anemia, including Hemorrhagic anemia due to excessive loss of blood, Hemolytic anemia due to red blood cell destruction and hematopoietic anemia due to less production of RBC. There are many causes of anemia, but the most common cause is iron deficiency (Irfan Ullah et al, 2014). Iron deficiency anemia lower the oxygen carrying capacity of blood with symptoms such as listlessness, disturbance, behavioral and impaired performance in some cognitive works (Underwood, 2001). Moreover, iron deficiency is often correlated with unusual tiredness, breath shortness, decreased physical performance and learning disability in children (Clark, 2008). Further, immune functioning is also dependent on the proper intake of iron (Zheng et al., 2008). Most frequently amount of iron deficiency anemia occurs in adolescence because of excessive physical growth in both genders, especially in young girl’s menstruation and dieting for fear of obesity in teenage. Both poor and socially advanced countries face this problem (Dallman et al).
Adolescent nutrition is decisive for proper growth and development and a thing that is required for achieving full developmental potential. Suboptimal nutrition may contribute to delayed and stunted growth as well as impaired development. As adolescents undergo a period of rapid growth and development, adequate nutrient intake of both( macro and micronutrients) is critical. Many of the risk factors that impact maternal and newborn health exist right from adolescence, including nutritional deficiencies. Pre pregnancy wasting in adolescents is usually reflected as a low body mass index (BMI ; 18.5). Low BMI significantly increases prenatal risks, including stillbirths, preterm births, and small for gestational age, and low birth weight (LBW) babies. Iron deficiency anemia is among the top 10 causes of disability-adjusted life years lost among adolescents. The concern is especially warranted for adolescent girls because their iron requirements are relatively high and because they may be on the cusp of motherhood. While most programs are targeted at pregnant women, the depletion of iron stores in women starts during adolescence with the onset of menstruation. More recently, there has been a growing interest in adolescent girls’ nutrition as a means to improve the health of women and children. Each year around 16 million babies are born to adolescent girls between the ages of 15 and 19 years, accounting for over 10% of the total births each year. Association of pregnancy of adolescent is occurring with greater risk to the mother and newborn, including anemia, mortality, stillbirths, and prematurity especially, since the adolescent girls are not physically maturing themselves (Rehana A et al 2016). Adolescence is characterized by a large growth spurt and, during this time period, iron needs increase dramatically in both genders as a result of the expansion of the total blood volume, the increase in lean body mass and the onset of menses in young female adolescence.
Fruit and vegetable consumption is associated with primary prevention of Anemia. Many of the health benefits associated with diets high in fruits and vegetables are attributed to their phytochemical content. Broadly categorizing, dietary iron sources can be classified into haem and non-haem dietary sources. Haem sources are generally iron rich dietary sources like organ meat and green leafy vegetables whilst non-haem sources reasoned of being deficient in iron availability and absorption haven’t gained much importance (de Oliveira Otto et al., 2012; Hurrell and Egli, 2010). The natural sources of iron are considered safe and healthy because they are better regulated, causing less damage to the body.

Vegetables are important constituents of the diet and provide significant quantities of nutrients, especially vitamins, minerals and fiber. Daily consumption of vegetables reduces the risk of cancer, heart disease, premature ageing, stress and fatigue primarily due to the integrated action of oxygen radical scavengers. One such vegetable available is carrot, which is an excellent source of carotene and antioxidants.
Pomegranate, Beetroot and carrot juices are very help full for treating anemia have a great source of vitamin C and iron content in combination have many anti antioxidant properties.
Pomegranate fruit from the tree Punica granatum has been used for centuries for medicinal purposes and is described as “nature’s power fruit”(Lei Wang et al, 2014). Current research has shown that pomegranate juice (PJ) or pomegranate extracts (PE) boost up the anemic cell to reproduce the red cell and increase the level of hemoglobin in adolescence.
For thousands of the years Pomegranate has been used to cure a great range of diseases across different civilizations and cultures. It has great nutritional values and numerous health benefits. By consuming this fruit in any form healthy blood flow can be maintained in the body. Seed extracts of pomegranate supplies iron of blood and thus, help to decrease the anemic symptoms, including fatigue, wooziness and weakness and hair loss (Neeru Agrawal, 2016). The other advantages of pomegranate fruit include reduction in the occurrence of premature infants and it is also beneficial for the expected mothers to avoid having low weight infants during birth. Regular intake of pomegranate consumed by elder people reduces the likelihood of creating Alzheimer’s disease. It helps in controlling aging issues, thereby sustaining the youthful and glowing skin. During the menopause period, consumption of pomegranate allows a woman to overcome from her depression interval (Stover et al, 2007).
The fruit is the major source of dietary pomegranate photochemical (Seeram NPetal 2006). Pomegranates may be consumed in a variety of ways; however, they are most popularly consumed as fresh fruit, beverages (juice), as food products (jams and jellies), and also as extracts in which they are used as botanical ingredients in herbal medicines and dietary supplements. The edible parts of the pomegranate fruit, which is approximately 50% of the total fruit weight, are comprised of 80% juice and 20% seeds (Aviram M et al, 2000). Fresh juice of pomegranate contains large amount of water almost 83% water, total sugars 10%, and the amount of pectin is 1.5% ,and ascorbic acid, polyphenolic and flavonoids also present. The seeds of the pomegranate are rich in crude fibers, pectin and sugars. Fructose and glucose are present in similar amounts in pomegranate juice (PJ) and 50% of its ash content is calcium. The principle amino acids in pomegranate juice are glutamic and aspartic acids (Aviram M et al, 2000).

Carrot juice has high nutritional value also has boosted properties of immunity, helps to heal minor wounds, injuries, overcome the risk of disease of heart and blood pressure. It excretes fat and bile from liver work as a cleanser for the liver. It also helps to reduce anemia, beneficial for eye health and overcome the risk of high blood pressure, stroke, heart diseases and some type of cancer. In ancient times, people have recognized as the special property of iron wrolstat et al, (2004). Carrot is a herbaceous plant containing about 87 percent of water and have many mineral salts and vitamins. Carrots has an excellent source of vitamin A, it also provide 6 percent of vitamin c and 2 percent of iron needs according to per servings. They are also a good source of potassium, vitamin B6, folic acid, thiamine and magnesium. Carrots contain fiber, vitamin k, phosphorous, vitamin E and zinc. Potassium which is present in carrots is an important component, controlling many functions of the cell such as electrolyte balance blood flow heart beat rate etc. carrots also have some other essential carb and oils. It is also well known for its healing power, sweating, antiemetic diuretic, rematerializing, and sedative properties (Donglin Zhang et, al).

Beetroot is also known to be very effective in fighting anemia. It is a vegetable that is filled with iron content. It has the property to reactivate and repair your red blood cells. Once the red blood cells are activated, the supply of oxygen to all parts of the body increases. Adding beet-root in any form in your daily diet will help to easily fight anemia.
Beetroot is a nutritious vegetable that is an ideal component of a healthy diet. It has a rich source of carbohydrates, a good source of protein and has high levels of important vitamins and minerals. It is a good source of dietary fiber and has practically no fat and no cholesterol. It supplies the principle antioxidants, which help to scavenge the free radicals. It also has several therapeutic properties like immune boosting; treating anemia, circulatory disorders, cancer, and alcohol induced liver failure, digestive disorders. Beetroot as vegetable are consumed raw as well as cooked. The growing nutritional awareness coupled with the consumer’s desire for a healthier lifestyle has greatly impacted the evolution of juice and juice based drinks. Hence fermented beetroot beverage has been developed to provide a refreshing, healthy and thirst quenching drink to our society and at the same time provides more health benefits ( Meghana Reddy et al, 2013). Currently, one of the more popular natural foods considered to help athletic performance is beet root, one of the most common varieties of beet in North America. Beetroot is an excellent source of antioxidants and micronutrients, including potassium, betaine, sodium, magnesium, vitamin C, and nitrate (NO3) and contains 29 kcal per 100 g. The color of beetroot stems from its purple and yellow pigments betacyanin and betaxanthin, respectively known collectively as betalains. These betalains have potential antioxidant capabilities. Interestingly, BRJ has been marketed on the Internet to support digestive and blood health, improve energy, be a natural cleanser, and increase levels of nitric oxide (NO) leading to increased blood flow. In addition, beet root juice has been indicated to possess anticancer properties, can lower the risk of coronary events, lower blood pressure, and reduce inflammation. These claims have boosted the popularity of beat root juice (Michael J,et al ,2013)
In developing countries Iron deficiency anemia is a major health problem. Anemia reduces physical work capacity and cognitive function and adversely affects learning and scholastic performance in schoolgirls entering adolescence. It results in increased morbidity from infections. A dose–response relationship between Hb (hemoglobin) and cognitive function in children with iron deficiency has been reported. Also, a possible association between depression and decreased ferritin level before the occurrence of anemia has been suggested. Iron deficiency anemia affects 40–50% of preschool and primary school children in Asia(Reza Akramipour et all,208).
The aim of present study was to assess the prevalence of anemia and its treatment with beetroot, pomegranate andcarrot juices, Iron deficiency and Iron deficiency anemia among adolescent school girls from a high school of Faisalabad. The Blood sample was obtained from each girl . Blood cell count (CBC) and differential white blood cell count were performed on the cell counter machine H1. Hemoglobin concentration was measured using the cyanmethhemoglobin method Serum ferritin level was determined using an immunoradiometric assay. Iron deficiency and IDA were defined as a serum ferritin (SF),20 mg/l and SF, 20 mg/l along with Hb,12 g/dl, respectively. Anemia was defined as an Hb, 100 g/day, lower levels may be considered. Maize flour: fortificant, ferrous fumarate at level, at least 5 mg/day of additional iron, but no ; 25 ppm (above current level available). Complementary foods and school programs: fortificant, ferrous sulfate + ascorbic acid or ferrous fumarate + ascorbic acid at level: based on specific requirements for age.

Pahwa et al.(2007) conducted a study and assessed the impact of double fortified salt (DFS) on iron and iodine status of adolescent girls(17-19 years).A sample size of 61 was randomly divided into an experimental group (n = 31) which was given DFS and control group (n = 30) which was given iodized salt for a period of 11 weeks. Eight subjects from the experimental group were dropped because of noncompliance to the use of DFS. Therefore, the study was completed by a total of 54 subjects. Baseline information like age, family size, amount of salt consumed etc. was collected. Anthropometric measurements like height, weight, mid upper arm circumference, body fat percentage, waist and hip circumference were taken and waist hip ratio and BMI were calculated at both pre and post intakes. Biochemical estimations for hemoglobin and urinary iodine excretion were carried out. The monthly salt consumption by the subject ranged from 1.0 – 2.2 kg. The diets of the majority of the subjects were found to be deficient in energy, protein, iron, thiamine, riboflavin, vitamin A and niacin. However, they met the RDAs for calcium, folic acid and ascorbic acid. No significant changes were seen in the anthropometric measurements after supplementation. A significant increase in the hemoglobin level of both the groups was seen, but the percentage increase was higher in the experimental group.

Nutrition education

Upadhyay et al. (2002) conducted a research in this research he observed the impact of combination of media vs. a single, on nutrition knowledge and hemoglobin status. After 60 days, a significant rise in post exposure knowledge value of both the groups was observed. Between the group’s multimedia group scored significantly higher than printed media group. Mean hemoglobin concentration was found to be higher, though non significant. Similarly, in the present study the subjects were exposed to short lectures, and other visual aids such as folder, flash cards, posters and display of raw foods which showed a significant rise in post exposure knowledge scores
Improving bioavailability of dietary iron

Bio availability of a nutrient particularly with reference to a trace mineral is defined as the proportion of a nutrient in food which is absorbed, transported to its site of action and converted to its biologically active form (O’Dell, 1984).Iron availability depends on iron exchange reactions in which legends’ of digestive tract act as donors and mucosal iron receptor protein as the acceptor. The rate and extent of this reaction depends on the number and activity of the mucosal iron receptor site determined physiologically by iron status of individual and chemical state of iron in the digestive mixture (Chidambaram et al., 1989).
Management of anemia through beetroot pomegranate and carrot juices
The improvement in hemoglobin level by using beetroot juice has been revealed by Priya et al., 2013. In this study adolescent school girls, in observed as experimental group. The objective of the study was to assess the effectiveness of beetroot juice on hemoglobin among adolescent girls. True applicable study design was adopted and the study conducted at Aringar Anna Government Higher Secondary School, Chennai, Tamilnadu. A total of 60 adolescent girls was selected for the study, in that 30 girls were in experimental and 30 girls were in the control group who fulfilled the inclusion criteria were selected by using simple random sampling technique. The freshly prepared beetroot juice was administered to the samples for 20 days in mid- morning. Pre and post assessment was done by using the checklist for assessing the signs and symptoms of anemia. The Current study also focused on the management of anemia with freshly prepared beetroot juice and monitoring the health status of adolescent girls.
Another study was conducted by Ghavan et al. (2016) for the evaluation of the effectiveness of beetroot to enhancing the level of HB in adolescent girls with anemia. For this study data were collected by using direct interviews, by using anemia checklists, a method for the confirmatory diagnosis and took a blood test to assess the correct level of hemoglobin, This study was conducted for 20 adolescent girls with iron deficiency anemia and herbal extract given for a period of 1month. Pre-test took before starting intervention and post-test at the end of the month. There was seen the significant achievement for the improvement in the level of HB. This study proves that the treatment with beet root will effective for improving the level of the hemoglobin. The Present study reflected that herbal extract has been playing a key role with iron deficiency anemia as demonstrated by improved hemoglobin and micro-nutrients level with fresh fruits and vegetable juices in the blood.
Carrot juice has the therapeutic property which can use for boosting of immunity, helps to heal minor wounds, injuries, overcome the risk of heart disease and blood pressure. It cleans the liver from excreting fats and bile, it helps to reduce anemia, beneficial for eye health, and overcome the risk of high blood pressure, stroke, heart disease and some types of cancer. In previous times, human has recognized the special properties of iron in health. Wrolstad et al, (2004).
For the confirmation of findings hemoglobin percentage Agrawal et al. (2016) conducted a study on pomegranate juice and with work 100 gm juice of Pomegranate was consumed daily in every morning. After every week of consumption, the Hb%age was determined by Sahli’sHemoglobinometer (Acid Hematin Method).The whole process was repeated thrice at the same time to get accuracy in results. To observe the actual effect of Pomegranate on rise of Hb%age the initial value of Hb was recorded just before starting consumption (at zero week) and also one week after stopping consumption of Pomegranate juice. Present study concluded that Pomegranate is proved to be useful for improving hemoglobin level in blood and thus preventing a person from “Anemia”.

(Ghawte et al, 2016) stated that Anemia is a problem in which level of Hemoglobin in blood the standard reference range appropriate for age and sex. Around 30% of total world population is anemic and half of these some 600 million people have iron deficiency. In Ayurveda , Anemia mentioned in Charaka Samhita, Yogaratnakara, Vagbhata in which skin, nails, eyes become fade called anemia. It can be correlated with Anemia. The clinical sign and symptoms of anemia reflects diminished oxygen supply to the tissues,there for skin become pale. According to researcher Menorrhagia is a common cause of anaemia in female. Pomegranate is selected for their wide spectrum action on Anemia like Hrdya (Good for Heart) , good source of Folate, Vitamin K, Vitamin C . To evaluate the effect of pomegranate in the treatment of iron deficiency anemia. Total 20 female patients of iron deficiency anemia with 4weeks duration were enrolled and randomly allocated to Group A (Oral pomegranate juice 50ml daily once at morning), Group B (Only Tablet Ferrous sulfate 100mg Daily Once) . Duration of treatment was considered about 1 month. Plasma ferritin, Hemoglobin, palpitation, Breathlessness, Paleness observed at weekly interval. With the treatment of 4 weeks, Group A showed dramatic improvements in signs and symptoms. Resolution of Plasma Ferritin , Hemoglobin, Palpitation, occurred in about 100% of patients. Whereas 90% of patients having reduction in Paleness and cessation of Breathlessness in 70%. In patients of Group B, anemia and Mean corpuscular volume shows good results in only 60%. Cessation of palpitation and paleness occurred in 50% whereas Hemoglobin elevetated in 70%. So use of Pomegranate Juice showed significant effect when compared before and after treatment by applying ‘t’ test to parameters, p