In Vitro Calcium Bioaccessbility in Moringaoleifera Vegetable Leaves: Potential Plant Food to Increase Dietary Calcium Intake in Developing Countries.

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Abstract

ISSA, JOSEPH YOHANE. In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable Leaves: Potential Plant Food to Increase Dietary Calcium Intake inDeveloping Countries. (Under the direction of Dr. Jonathan C Allen).

Low calcium intake, poor calcium absorption, exessive calcium losses, or some combination of these factors contribute to calcium deficiency diseases. Calcium deficiency can lead

osteoporosis, reduced bone mass, hypertension and colon cancer among other diseases.Calcium in bones plays a role in bone mineralization. In other body tissues it plays roles in mediating vascular contraction and vasodilation, muscle contraction, nerve

transmission, and glandular secretion.The best food sources of calcium include milk and dairy products, especially cheese and yogurt, and selected seafoods, such as salmon and

sardines (with bones), clams, and oysters. Bioavailability and bioaccessibility of calcium is high in milk and dairy products due to the presence of lactose and other factors. However, calcium bioavailability or bioaccessibility is low in most vegetable plants due to presence of

other substances like oxalic acid, phytate and other competing minerals.

Moringa oleifera, a tropical and sub-tropical vegetable plant, has a big potential to contibute a substantive portion of the recommended daily allowance (RDA) for calcium and reduce calcium deficiency diseases. The objective of this study was to determine the calcium

content, digestibility and bioaccessibility in the leaves of Moringa oleifera.

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bioaccessibility. The digestible and bioacessible calcium was measured using AAS after an

in-vitro digestion and an in-vitro absorption processes respectively.The results indicated that

Moringa oleifera had higher percentage of calcium than spinach and sweet potato leaves. The mean percentage calcium was 1.67±0.0014, 1.44±0.0014 and 1.55±0.00 for Moringa oleifera

samples from India (M3), from Malawi (M4) and another sample from Malawi (M5) respectively. The mean percentage calcium in spinach and sweet potato leaves were 0.99±0.0007, and 1.06± 0.0014 respectively.The mean percentage digested calcium was

21.68±2.65, 40.48±3.42 and 37.24±1.66 for Moringa oleifera samples from India (M3), from Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The mean percentage digested calcium in spinach and sweet potato leaves were 1.63±0.08, and 3.01± 0.68 respectively. The mean percentage dialyzed or bioaccessible calcium was 6.82±1.80, 10.75±0.96 and 11.15 ±2.88 for Moringa oleifera samples from India (M3), from Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The mean percentage dialyzed calcium in spinach and sweet potato leaves were 0.55±0.37, and

3.08±2.05 respectively.

The results showed that there was a statistically significant different in percentage calcium

content from the Moringa oleifera samples from India and the two samples from Malawi. The digested calcium in the two Moringa oleifera samples from Malawi was found to be statistically similar; however both samples from Malawi were statistically different from

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In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable Leaves: Potential Plant Food to Increase Dietary Calcium Intake in Developing Countries

by Joseph Yohane Issa

A thesis submitted to the Graduate Faculty of North Carolina State University

in partial fulfillment of the requirements for the degree of

Master of Science

Food Science

Raleigh, North Carolina 2012

APPROVED BY:

________________________ ___________________________ G.Keith Harris, Ph.D. Rick Brandenburg, Ph.D.

________________________________ Jonathan C. Allen, Ph.D.

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DEDICATION

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BIOGRAPHY

Joseph Issa was born on 5th of January 1982 in Blantyre, Malawi. He grew up in Malawi and did his secondary school education at Chiradzulu secondary school in the same country. He pursued his bachelor’s degree in Education Science at Chancellor College, a constituent

college of the University of Malawi. Joseph graduated with a Bachelor’s degree in May 2006 with a major in Chemistry and minor in Biology.

After graduating from the University of Malawi, Joseph worked in the Ministry of Education,

Malawi where he worked as a secondary school teacher for the sciences. He worked as a teacher from June 2006 to January 2007. Thereafter he joined the Malawi Industrial Research

and Technology Development Centre (MIRTDC) in February 2007. At MIRTDC, Joseph worked as a Chemical scientist.

While at MIRTDC, Joseph got a scholarship from the United States Agency for International

Development (USAID) to study for a Masters degree in July 2009. Joseph pursued a Master of Science degree in Food Science at North Carolina State University under the guidance of

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ACKNOWLEDGEMENTS

I thank all my Lecturers, for the support and encouragement given to me during my study at North Carolina State University. Many thanks should go to my Advisor and committee chair of my thesis research, Dr. Jonathan Allen. I thank Dr. Allen for all the support, encouragement and guidance that he gave me during my thesis research work. Thanks should also go to all my committee members, Dr Rick Brandenburg and Dr Keith Harris for their guidance and mentorship. Thanks to Ruth Watkins, the laboratory manager of the Nutrition Technical Services Lab, Food, Bioprocesing and Nutrition Sciences Department at NC State University and Mr. Weiting Cai for their assistance and guidance in conducting my laboratory analysis.

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TABLE OF CONTENTS

LIST OF TABLES ...vii

LIST OF FIGURES ... viii

CHAPTER 1-LITERATURE REVIEW Introduction ... 1

Calcium sources and functions ... 1

Calcium bioavailability and absorption ... 5

Effects of dietary components on calcium absorption ... 6

Calcium and vitamin D ... 8

Effects of calcium deficiency ... 10

Nutritional needs of calcium ... 11

Calcium toxicity ... 12

Techniques for measuring calcium bioavailability ... 13

In-vivo methods ... 13

In-vitro methods ... 15

References ... 16

BIOACCESSBILITY OF CALCIUM IN MORINGA OLEIFERA LEAVES Introduction ... 23

History and use of Moringa oleifera ... 23

Moringa oleifera nutrition ... 25

Materials and Method ... 26

Sample collection and preparation ... 26

Sample collection ... 26

Sample preparation ... 26

Evaluation of total calcium ... 27

Evaluation of calcium digestibility ... 27

Gastric digestion ... 29

Intestinal digestion ... 29

Evaluation of calcium bioaccessibility ... 29

Calculation of calcium bioaccessibility ... 31

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Results ... 32

Calcium content ... 32

Calcium digestibility ... 34

Calcium bioaccessibility ... 35

Discussion ... 37

Conclusion ... 41

References ... 42

APPENDICES ... 44

Appendix 1. SAS output data for percentage Calcium content in M3, M4 and M5 ... 45

Appendix 2. SAS output data for percentage digested calcium in M3, M4 and M5 ... 48

Appendix 3. SAS output data for percentage dialyzed calcium in M3, M4 and M5 ... 51

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LIST OF TABLES

Table 1.1 Calcium Dietary Reference Intakes (DRIs) for Adequacy

(Amount/day) ... 4 Table 1.2 Effects of dietary components on calcium absorption ... 7 Table 2.1 Nutrient comparison of Moringa oleifera leaves with other foods,

per 100g ... 19 Table 2.2 Total calcium content from Moringa oleifera, spinach and sweet

Potato leaves ... 27 Table 2.3 Calcium digestibility from Moringa oleifera, spinach and sweet

potato leaves ... 28 Table 2.4 Calcium inaccessibility from Moringa oleifera, spinach and sweet

potato leaves ... 31

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LIST OF FIGURES

LITERATURE REVIEW

Figure 1. Production, metabolism and calcium homeostasis regulation of vitamin D... 5 MATERIALS AND METHODS

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CHAPTER 1

LITERATURE REVIEW

JOSEPH YOHANE ISSA

DEPARTMENT OF FOODBIOPROCESSING AND NUTRITION SCIENCES

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Introduction

1.1Calcium sources and Functions

Calcium is a very important and abundant mineral in the body and accounts for one to two percent of the body weight. More than 99 percent of the body calcium is found in bones and teeth, the rest is found in blood, muscle, extracellular fluid and other body tissues (Gropper,

2009).In bone, calcium exists primarily in the form of hydroxyapatite (Ca10(PO4)6(OH)2), and

bone mineral is almost 40 percent of the weight of bone (IOM, 1997). Calcium in bones play

a role in bone mineralization, in other body tissues it plays roles in mediating vascular contraction and vasodilation, muscle contraction, nerve transmission, and glandular

secretion. Bone is a dynamic tissue that continuously undergoes osteoclastic bone resorption and osteoblastic bone formation.

In the body, calcium is absorbed by active transport and passive diffusion across the

intestinal mucosa. Active transport of calcium into enterocytes and out on the serosal side depends on the action of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin

D, and its intestinal receptors. However, passive diffusion involves the movement of calcium between mucosal cells and is dependent on the luminal:serosal calcium concentration

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Fractional calcium absorption varies through the lifespan; it is highest (about 60 percent) in infancy (Abrams et al., 1997a) and rises again in early puberty. Calcium excretion from the

kidneys depends on the filtered load and the efficiency of reabsorption where the latter is regulated primarily by the PTH level.Calcium metabolism differs from one race to

another.Parathyroid Hormone (PTH), calcitonin, and vitamin D regulatecalcium metabolism to maintain serum calcium level. Low intake of calcium leads to release of calcium from bone, thereby increasing the risk of osteoporosis (Bendich, 2001).The best food sources of

calcium include milk and dairy products, especially cheese and yogurt, and selected seafoods, such as salmon and sardines (with bones), clams, and oysters (Gropper et al.,

2009). Bioavailability of calcium from nonfood sources, or supplements, depends on the presence or absence of a meal and the size of the dose. Weight-bearing physical activity or mechanical loading determines the strength, shape, and mass of bone. Although exercise and

calcium intake both influence bone mass, it is unclear whether calcium intake influences the degree of benefit derived from exercise. There is insufficient evidence to justify different

calcium intake recommendations for people with different levels of physical activity. Calcium can be obtained from calcium-rich foods such as milk, dairy products and other

fortified foods such as cereal and juice beverages (IOM, 1997). In other countries however, including USA and Malawi, milk consumption is limited due to the undesirable effects of lactose intolerance (Scrimshaw and Murry, 1988). The Recommended Dietary Allowances

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Inadequate calcium consumption may have an adverse effect of reducing blood calcium level, resulting in development of tetany, and can lead to respiratory and cardiac failure as a

result of impaired muscle function (Luke, 1984). Increased calcium intake can lower the risks of systolic and diastolic blood pressure, thereby providing some protection against

hypertension (Bucher et al.,1996). About half of calcium in the blood exists in form of free dissolved calcium ion (Ca2+), about 40% is bound to proteins, mainly albumin and prealbumin, and the remaining percentage (up to 10%) is in complexes with sulphate,

phosphate or citrate (Muban, et al., 1996). Calcium helps to initiate the changes needed for the formation of blood clots by binding to fibrin and other proteins in the clotting cascade.

Thrombin requires calcium to help in the polymerization of fibrinogen to fibrin (Czajka-Narins D M, 1992). Calcium is needed for transmitting nerve impulses. Calcium makes acetylcholine available for nerve impulse transmission whereby acetylcholine is required to

pass messages from one nerve cell to the adjacent one. Calcium influences the transmission of ions across membranes of cell organelles, the release of neurotransmitters at synaptic

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Table 1.1:Calcium Dietary Reference Intakes (DRIs) for Adequacy (Amount/day)

Life Stage Group AI EAR RDA

Infants

0 to 6 mo 200 mg — — 6 to 12 mo 260 mg — —

Children

1–3 y — 500 mg 700 mg 4–8 y — 800 mg 1,000 mg

Males

9–13 y — 1,100 mg 1,300 mg 14–18 y — 1,100 mg 1,300 mg 19–30 y — 800 mg 1,000 mg 31–50 y — 800 mg 1,000 mg 51–70 y — 800 mg 1,000 mg > 70 y — 1,000 mg 1,200 mg

Females

9–13 y — 1,100 mg 1,300 mg 14–18 y — 1,100 mg 1,300 mg 19–30 y — 800 mg 1,000 mg 31–50 y — 800 mg 1,000 mg 51–70 y — 1,000 mg 1,200 mg > 70 y — 1,000 mg 1,200 mg

Pregnancy

14–18 y — 1,100 mg 1,300 mg 19–30 y — 800 mg 1,000 mg 31–50 y — 800 mg 1,000 mg

Lactation

14–18 y — 1,100 mg 1,300 mg 19–30 y — 800 mg 1,000 mg 31–50 y — 800 mg 1,000 mg

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1.2.1 Calcium Bioavailability and Absorption

Bioavailability for dietary supplements is defined as the proportion of a substance capable of

being absorbed and available for use or storage in the body (Srinivasan, 2001). Bioacessibility is the potential of a nutrient to interact with (and be absorbed by) an

organism. There are many chemical structures of calcium in food; however its bioavailability varies from one source to another. Calcium may be poorly absorbed from foods rich in

oxalic acid (spinach, sweet potatoes, rhubarb, and beans) or phytic acid (unleavened bread, raw beans, seeds, nuts and grains, and soy isolates). Some nutrients such as sodium, protein and caffeine have a great impact on calcium absorption and retention. High sodium chloride

intake results in increased absorbed sodium, increased urinary sodium, and an increased obligatory loss of urinary calcium (Kurtz et al., 1987). Protein increases urinary calcium

excretion, but its effect on calcium retention is not well described. However, available evidence does not warrant adjusting calcium intake recommendations based on dietary protein intake. Caffeine has a negative impact on calcium retention and has been associated

with increased hip fracture risk in women (Kiel et al., 1990).Conditions that produce lower levels of circulating estrogen alter calcium homeostasis; lower levels of estrogen are

associated with decreased calcium absorption efficiency. However, from available evidence, the calcium intake requirement for women does not appear to change acutely with menopause. Lactose-intolerant individuals absorb calcium normally from milk; however they

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In addition, consumption of vegetarian diets may influence the calcium requirement because of their relatively high contents of oxalate and phytate, compounds that reduce calcium

bioavailability. Calcium is transported from the intestinal lumen into the body mainly by an active transport mechanism. Active transport requires energy, involves a calcium binding

protein (calbindin) and is regulated by vitamin D3 (calcitriol). Vitamin D3 calcium dependent

transport is stimulated with the ingestion of low calcium diets, and conditions of growth, pregnancy and lactation in which calcium requirements are increased (National Research

Council, 1989). Calcium is also absorbed in the body via a passive transport mechanism. Absorption of calcium is increased via passive transport if there is an increased intake of the

mineral (National Research Council, 1989).

1.2.2 Effects of dietary components on calcium absorption

Lactose has been reported to increase calcium absorption and is possibly why calcium from

milk is highly bioavailable. The lactose-calcium complex prevents the precipitation of calcium in an insoluble complex as the contents of the intestinal tract changes from acid to

alkaline (Kocian et al., 1973).Vitamin D regulates the synthesis of a calcium binding protein that serves as calcium carrier in the intestinal cell. Increased concentration of the active form of vitamin D results in a 10 to 30% increase in calcium absorption (Burton, 1976). Insoluble

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Processing methods such as dehulling, soaking, ordinary cooking, pressure cooking and germination have been found to beneficially lower phytic acid content and improve the

bioavailability of minerals(Bishnoiet al., 1994). Oxalic acid binds with Ca, Fe and Mg, making these minerals unavailable for absorption. Generally, oxalic acid is highest in the

leaves, higher in the seeds and the lowest in the stems (Osweileret al., 1985).

Table 1.2: Effects of dietary components on calcium absorption

Inhibitors Promoters No effect

Phytate Lactose Phosphorus

Fiber Vitamin D Protein

Oxalate Ascorbic/ citric acids

Alcohol Pectin

Uronic acid

Cellulose

Sodium arginate

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1.2.3 Calcium and Vitamin D

The active form of vitamin D, 1,25 dihydroxy-vitamin D3 (calcitriol), regulates blood

calcium by increasing absorption of calcium through the small intestine, reducing calcium excretion through the kidney and regulating calcium deposition in the bones (Wardlaw,

1999).

The intestinal calcium absorption is mainly regulated by vitamin Dwhen increases in 1,25 dihydroxy-vitamin D increase active transport. The main biological function of vitamin D3

on regulators of calcium metabolism are stimulating the absorption of calcium from food across the intestine and participating in the incorporation of the absorbed calcium into the

skeleton (Norman, 1979). Adequate amounts of calcium and vitamin D throughout life helps reduce the risk of osteoporosis and reduces risk of fractures and falls (Calvo et al., 2005 and Iwamoto et al., 2004). Vitamin D deficiency causes poor bone mineralization of the collagen matrix in young children’s bones leading to growth retardation and bone deformities known

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1.2.4 Effects of calcium deficiency

Inadequate calcium intake, poor calcium absorption, exessive calcium losses, or some

combination of these factors contributes to calcium deficiency (Gropper, et al.,2009). Calcium deficiency is associated with many chronic diseases. In the United States, 10 million

people have osteoporosis and another 18 million are at increased risk; 80% of those affected are women (Bronner, 1992). Osteoporosis is a disease characterized by low bone mass and

structural detoriation of bone tissue, leading to bone fragility and increased susceptability to fractures, especially of the hip, spine and wrist (Kanis, et al., 1994). Ingestion of diets rich in calcium and vitamin D, weight bearing exercise, a healthy life style with no smoking or

excessive alcohol use can reduce the risks of developing osteoporosis(Riggs, et al., 1992; Vuori, 1996). Inadequate amounts of calcium and phosphorus coupled with insufficient

activity of vitamin D results in failure of bones to mineralize properly and bones weaken and soften, causing rickets in young children and osteomalacia in adults. Osteomalacia may cause fractures in the hip and spine bones while rickets can result into bowed bones, curved bones

and lumpy joints. Severe loss of calcium and phosphorus may lead to formation of cysts in the bones (Rasmussen, et al., 2000).

Research indicates that low calcium intake increases the risk of high blood pressure (Miller et

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It has been found that calcium may inhibit the formation of colon cancer cells by binding with bile acids and fatty acids, both of which can trigger abnormal cell growth within the

colon (Lupton, et al., 1996; Holtet al., 1998).

1.2.5 Nutritional needs for calcium

The nutritional requirements for people varies with age and activity. The dietary reference

intakes may vary in different countries. In America, the Dietary Reference Intakes (DRI) establish and Adequate Intake (AI) at 200mg/day and 260mg/day for infants aged 0-6 months

and 7-12 months, respectively. For children aged 1-3years and 4-8 years the DRIs set a Recommended Dietary Alllowance (RDA) at 700mg/day and 100mg/day, respectively, based

in an Estimated Average Requiremetn (EAR) of 500 mg/day and 800 mg/day.The calcium RDAs for both males and females in America are 1300mg/day, 1000mg/day and 1200mg/day for ages 9-18years, 19-70years and above 70 years respectively. The RDAs for pregnant and

lactating women are 1300mg/day and 1000mg/day for women aged 14-18years and 19-50years respectively(IOM., 2011).According to a study done in Malawi, a developing

country, food eaten at main meals, dinner and lunch contributed the largest proportions on energy, protein and calcium intakes (Hallund, etal., 2008).The study however showed that calcium intakes from women in the study was lower than the recommended intakes with

average intakes of 620mg/day and 622mg/day for non-lactating women and lactating women respectively.These values are far lower than the recommended intakes of 1000-1300mg/day

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The study was conducted between the months of March and May (the peak season for many fruits and vegetables) and fruits and vegetables contributed more to calcium intake.A nutrient

intake study among a longitudinal cohort of black South African children at four inceptions (from birth to ten years) found that calcium intake fell below the recommended intakes at all

the inceptions (Mackeown et al., 2003). During the four inceptions; 70%, 90%, 77% and 90% of the children under study were below the RDA at the first (1995), second (1997), third (1999) and fourth (2000) inception respectively.Another study on nutrient intake was

conducted in South Africa in three ethnic groups (black, white and mixed ancestry subjects) and found that mean dietary calcium was higher in white subjects than in black and mixed

ancestry subjects; however, calcium intake was low in all the groups with about half of the DRI of 1000mg/day (Charlton et al., 2005).In black, mixed ancestry, and white subjects, 84.4%, 88.4% and 78.6% respectively had inadequate (<67% DRI) calcium intakes.

1.2.6 Calcium Toxicity

Intakes of calcium of up to 2,500mg per day appears to be safe for most people (Gropper, et

al., 2009). There is no documented information on calcium overconsumption from food, as opposed to from dietary supplements. Overconsumption of calcium lead to elevated blood calcium (hypercalcemia) which results in loss of appetite, nausea, vomiting, constipation,

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People with hypercalciuria (urinary calcium intake levels of more than 4mg/kg body weight per day), or who ingest large amounts of calcium, are at the risk of developing

calcium-containing kidney stones (Gropper, et al., 2009).

The tolerable upper intake level (UL) of calcium has been recommended for people aged one

to eight years old and 19 to 50 years old to be 2500mg/day, for 9 to 18 years old, 3000 mg/day, and over 50, 2000 mg/day (IOM, 2011).

1.2.7 Techniques for Measuring Calcium bioavailability

Bioavailability of nutrients is either estimated by in-vivo or in-vitro experimental studies. In-vivo studies use animals as models for human calcium absorption.In-vitro studies use solubility or dialysability tests to simulate the conditions in the human stomach and intestines. Compared to in-vitro experiments, in-vivo experiments are time consuming, very expensive and often give variable results that are difficult to interpret. In in-vivo studies, laboratory animals are used as models for humans. In-vitro studies are popular nowadays because of their simplicity, accuracy, speed of analysis and relatively low cost (Wolter, et al.,

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1.2.7.1In-vivo methods

In-vivo studies are balance studies, which include chemical balances, radioactive balances and stable isotope balances. Balance studies have been conducted in both animals and humans in order to measure the apparent absorption of calcium from various diets, and under

different physiological conditions (Allen, 1982).

The difference between calcium intake and fecal calcium is defined as apparent absorption. Radioactive balance studies correct for endogenous excretion to determine true absorption.

Chemical balance studies do not expose subjects to ionizing radiation hence giving it preference as compared to other balance methods when human subjects are involved. Errors

when determining either intake or excretion can result in significant errors in absorption estimates (Hegsted, 1973).

There are a number of methods that use calcium isotopes; with those commonly used being radioactive such as 45Ca and 47Ca, however 47Ca is frequently used in humans because of its shorter half-life (Allen, 1982). To estimate calcium absorption using isotopes of calcium, the

difference between the intake and fecal output of the isotope is assumed to be the true absorption. There are two types of calcium isotope techniques; namely single-isotope and

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However the disadvantage is that if used in humans, the subjects are exposed to ionizing radiation, which may be unadvisable to some target groups such as pregnant women, infants

and young children (Layrisse, et al., 1990). Apart from radioactive isotopes, stable isotopes of calcium have also been used in different bioavailability studies. Use of these isotopes

employs the same mathematical techniques as in the other balance studies, but different instrumentation for measurement. Stable isotopes have an advantage over the radioactive isotopes because they have no health risks from ionizing radiation, do not decay and can be

freely used in food samples or food processing equipment without causing radioactive contamination. Stable isotope technique is however disadvantageous in that it is more

expensive than radioactive techniques (Weaver, 1988).

1.2.7.2In-vitro methods

Some in-vitro methods involve determining solubility of trace elements in different solutions whereas some methods involve extraction of the trace elements with chelating agents. Many

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This method has been the basis for many in-vitro methods for estimating the bioavailability of iron and other minerals including calcium. Miller’s in-vitro method used equilibrium dialysis of minerals and trace elements across a semi-permeable membrane as a model for the passage across the walls of the intestines. The assumption here is that the minerals and trace

elements that are dialyzable are available for absorption in the small intestines.

Calcium absorption in infants is mainly through passive diffusion (Barltrop et al., 1977, and

Bronner, 1992), however in adults it is mainly absorbed through an active transport mechanism and passive transport only when the intake has been increased to saturation. Some improved in-vitro dialysis methods have been developed to continuously remove the dialyzable components (Minihane et al., 1993).Minihane et al., (1993) used an Amicon stirred cell for continuous dialysis and the pH was gradually adjusted over 30 minutes period

to 7 before dialysis. The results indicated that the gradual change of pH during dialysis is very important in evaluating calcium bioavailability.

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J Nutr 131;4:1349S-50S.

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Weaver C (1988) Isotopic tracer methodology: Potential in mineral nutrition. In: Smith

KT.ed, Trace minerals in foods. New York: Marcel Dekker:429-54.

Wolter MGE, Schreuder HAW, Heurel GVD, Lonkhuijsen HIV, Hermus RIJ, (1993) A continuous in-vitro method for estimation of the bioavailability of minerals and trace elements in foods: Application to breads varying in phytic acid content. Brit J Nutr

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CHAPTER 2

BIOACCESSBILITY OF CALCIUM IN MORINGA OLEIFERA LEAVES

JOSEPH YOHANE ISSA

DEPARTMENT OF FOOD, BIOPROCESSING, AND NUTRITION SCIENCES

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2.1Introduction

2.1.1 History and uses of Moringa oleifera

Moringa oleifera is the best known of the thirteen species of the genus Moringacae. Moringa oleifera was originated from India in some 5,000 years ago. Moringa is mainly grown in the tropical and sub-tropical regions of the world. Moringa grows well in the dry sandy soils but it also tolerates poor soils, including coastal areas. In the tropical regions of Asia, Africa and South America, Moringa leaves are eaten fresh by the local people. The leaves, fruits, flowers and immature pods of this tree are edible and they form a part of traditional diets in many countries of the tropics and sub-tropics (Siddhuraju et al., 2003). Apart from being

used for nutritional benefits, M. oleifera has a great potential as a medicinal plant (Ferreira et al., 2008). The flowers, leaves and roots are used for the treatment of ascites, rheumatism and venomous bites and as cardiac and circulatory stimulants in folk remedies (Anwar et al.,

2007). As a traditional food plant in Africa, M. oleifera vegetable has potential to improve nutrition, boost food security, foster rural development, and support sustainable land care. In

some parts of the world such as Senegal and Haiti, health workers have been treating malnutrition in small children, pregnant and nursing women with Moringa leaf powder (Price, 1985). An 8-g serving of dried leaf powder supplies a substantial portion of the daily

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2.1.2 Moringa oleifera Nutrition

M. Oleifera has been reported in both scientific and popular literature as having numerous nutritional qualities. The leaves of M. oleifera are a good source of protein, vitamin A, B and C and minerals such as calcium and iron (Dahot, 1988).According to Trees for Life Organization, ―ounce for ounce, Moringa leaves contain more vitamin A than carrots, more calcium than in milk; more iron than spinach, more potassium than bananas, more vitamin C than oranges,‖ and the protein quality of Moringa leaves rivals that of milk and eggs.

Table 2.1: Nutrient comparison of M. oleifera leaves with other foods, per 100g.

Moringa oleifera: Natural Nutrition for the Tropics by Lowell Fuglie.

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A research study on nutritional and functional properties of M. oleifera leaves by Yang et al., (2006), found that fresh mature leaves and fresh young shoots had 454±63mg and

82±31mg of calcium per 100g of sample respectively. In their previous study on in-vitro iron bioavailability, (Yung et al., 2002) found that boiling the fresh leaves and dried powder of M. oleifera enhanced the in-vitro iron bioavailability by 3.5 and 3 times respectively. The review of literature has shown evidence that there is high calcium content in M. oleifera plant materials; however there are no data on the bioavailability of calcium from the plant

materials. Many plants contain components such as dietary fiber and organic acids that limit calcium solubility and accessibility to digestive and absorptive processes. This research was

therefore designed to investigate the calcium bioavailability in M. oleifera plant leaves by measuring bioaccessibility using an in vitro simulated digestive system.

2.2Materials and Methods

Study Design.

The study on calcium bioaccessibility in M. oleifera plant materials was divided into four concepts as shown below.

1) Collection and preparation of samples.

2) Measurement of total calcium

3) Evaluation of calcium in-vitro digestibility

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True bioavailability cannot be determined from this in vitro system because it does not distinguish between forms of calcium that can be absorbed by regulated active transport

processes and passive absorption, nor between retention and rapid renal excretion.

2.3.1 Sample collection and preparation

2.3.1.1 Sample collection

Green and mature Moringa leaf samples were collected from several trees (about 3 trees in each area) in low lying hot areas of Malawi where it grows naturally and the conditions are favorable for its growth. In these areas, average temperatures are 30 degrees Celsius. The temperature can reach up to 40 degrees Celsius in the dry season. The sample collection

areas, Lilongwe and Karonga, are along the lake shore of Lake Malawi and Shire River. These areas are generally hot and flat with sandy loam soils.

Some Moringa oleifera samples were bought in USA from an Internet distributor (MoringaSource.com). These commercial product samples were originally brought from India and sold in the United States. These powdered Moringa oleifara leaf samples were packed in aluminum foiled bags.

2.3.1.2 Sample preparation

Leaf samples from Malawi were air dried, pounded in a mortar into powder, packed in plastic bags and transferred to the Nutrition Technical Services Laboratory at North Carolina State University in USA for analysis. Commercial samples from India were also air dried, pounded

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2.3.2 Evaluation of Total Calcium

Total calcium from the samples was determined by an atomic absorption spectrophotometer

(AAS). Powdered dry leaf samples were subjected to dry-ashing before the determination of calcium. A 0.1-g sample of each material was weighed and ashed at 525°C for 8hours. The ash was dissolved with 0.1% HCl and 0.5% lanthanum oxide (La2O3) followed by AAS

analysis to determine calcium content.

2.3.4 Evaluation of Calcium Digestibility

In order to determine calcium digestibility, in-vitro digestion was used. The Moringa oleifera

leaf powder was subjected to a simulated gastric phase followed by intestinal phase of

digestion. Pepsin enzyme from porcine gastric mucosa (Pcode 1000848645 Sigma-Aldrich, St. Louis, MO, USA) and hydrochloric acid was used in the gastric phase digestion. This was followed by pancreatic enzymes (pancreatin from porcine pancreas, P1750, Sigma-Aldrich)

and porcine bile extract (B8631, Sigma Aldrich) solution in the intestinal phase digestion. This method followed simulated digestion conditions.Figure1 below shows a flow diagram

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1) Gastric digestion

Dissolve 0.2 g pepsin into 5 ml 0.1 N HCl

Measure the sample needed for test (0.1g).

Add 0.25 ml of the pepsin solution to the sample;

Adjust pH to 2.0 with HCl;

Incubate in a shaking water bath at 37 °C for 120 strokes per minute for 2 hours;

Place on ice for 10 minutes;

Raise pH to 7 by adding 1 M NaHCO3

2) Intestinal digestion

Dissolve 0.05 g pancreatin and 0.3 g bile extract in 25 mL 0.1 M NaHCO3

Add 1.25 mL of the pancreatin, bile extract, and lipase solution to each sample;

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Place on ice for 10 minutes;

Adjust pH to 7.2 by addition of 0.5 M NaOH;

Bring to a common volume for all samples;

Filter the solutions under normal condition and analyze the filtrate of each sample by atomic absorption for calcium digested.

2.3.5 Gastric Digestion

Samples (0.1 g) each were measured and added to a 0.25 mL of pepsin solution in the digestion tubes. The pH of the samples was then adjusted to 2 by the addition of 6 N HCl.

The samples were then incubated in a shaking water bath at 37 °C for 2 hours in order to simulate the digestion process in the stomach. The samples were then placed on an ice bath

for 10 minutes in order to stop the pepsin digestion and then its pH was raised to 7 by adding NaHCO3 to prepare it for intestinal digestion.

2.3.6 Intestinal Digestion

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Thereafter, samples were incubated in a shaking water bath at 37 °C for 2 hours and then placed on an ice bath for 10 minutes to stop the intestinal digestion. The pH of the samples

was adjusted to 7.2 by the addition of 0.5 M NaOH. Samples were then brought to a common volume by addition of distilled de-ionized water, filtered under normal conditions and the

filtrate of each sample was analyzed for digested calcium by an atomic absorption spectrometer.

2.3.7 Evaluation of Calcium Bioaccessibility

The bioaccessibility of calcium (defined as the low-molecular weigh soluble fraction in gastrointestinal fluids) in the samples was determined by simulated in-vitro absorption of calcium by using dialysis tubes. This involved the passive trans-membrane diffusion of calcium ions from one medium to another. A 10-mL portion of each sample (previously

digested) was loaded to the inside of dialysis tubing by pipettes. The outside compartment of the dialysis tubing of each sample was loaded with 25 mL solution of 0.9% NaCl with albumin. The samples were then dialyzed at room temperature for 24 hours. The volumes of

the solution outside and inside the dialysis tubing were measured and emptied in separate test tubes for calcium analysis. Calcium was analyzed by atomic absorption and the calcium that

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Load 10 mL(previously digested material) sample to the inside of the dialysis tubing carefully by using a pipette;

Slowly withdraw the pipette as dispensing;

Fill the outside of the Floatalyzer™ G2 apparatus (Spectrum Laboratories, Inc., Rancho Dominquez, CA) with 25 mL solution of 0.9% NaCl with 0.1% bovine serum albumin;

Screw the cap back in place to create a closed seal;

Dialyze at room temperature for 24 hours;

Measure the volumes inside and outside of the dialysis tubing after dialysis;

Transfer contents inside and outside of the dialysis tubing into separate test tubes for calcium analysis;

Run atomic absorption analysis for calcium in the solutions

Figure 2. A diagram for an in-vitro calcium bioaccessibility.

2.3.8 Calculation of calcium bioaccessibility

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2.3.9 Data Analysis

SAS software (Cary, NC) was used to statistically analyze the data. This software was used

to see the relationship between the Moringa oleifera samples from Malawi and the other from India. It was also used to see the mean differences between Moringa oleifera, sweet potato leaves, and spinach in terms of calcium content, digestibility and bioaccessibility. Tukey’s Studentized Range (TSR) test was used to determine whether or not significant

differences existed in the mean values of the sample calcium content, digestibility and bioaccessbility at P ≤ 0.05.

2.4 Results

The results were categorized into three groups; percentage calcium content, percentage digested calcium and percentage dialyzed calcium.

2.4.1 Calcium content

Calcium content of Moringa oleifera samples and the control samples, spinach and sweet potato leaves are provided in Table 2.2

Calcium from the samples was expressed as percentage of the dry matter. The results of

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However, among the three Moringa oleifera samples, there was a significant difference in the percentage calcium content (P< 0.05) using Tukey’s Studentized Range Test at α=0.05.

The mean percentage calcium was 1.67 ± 0.0014, 1.44 ± 0.0014 and 1.55 ± 0.00 for Moringa oleifera samples from India (M3), from Karonga, Malawi (M4) and another sample from Lilongwe, Malawi (M5) respectively. The mean percentage calcium in spinach and sweet potato leaves were 0.99 ± 0.0007, and 1.06 ± 0.0014 respectively.

Table 2.2: Total calcium content from Moringa oleifera, spinach and sweet potato leaves (mean ± standard deviation of 2-4 replicates per sample).

Sample1 Total calcium content (%) N replicates

M3 1.67 ± 0.0014 3

M4 1.44 ± 0.0044 4

M5 1.55 ± 0.00 2

S 0.99 ± 0.0007 2

SP 1.06 ± 0.0014 2

1

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2.4.2 Calcium Digestibility

The digested calcium from Moringa oleifera samples and the control samples, spinach and sweet potato leaves are provided in Table 2.3.

Digestibility of calcium from the samples was expressed as percentage of the calcium in dry matter that was solubilized by the digestion procedure. The digestibility results were found

from the three Moringa oleifera samples and the control samples; spinach and sweet potato leaves. The entire digestion procedure was replicated three times, using triplicate samples in

each replicate. The results indicated that Moringa oleifera samples had higher digested calcium than spinach and sweet potato leaves. Using Tukey’s Studentized Range Test at

α=0.05, there was no significant difference in total digested calcium between the two samples

from Malawi, M4 and M5. However, there was a significant difference in percentage digested calcium between Moringa oleifera samples from India and Malawi (P < 0.05). The mean percentage digested calcium was 21.68 ± 2.65, 40.48 ± 3.42 and 37.24 ± 1.66% for

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Table 2.3: Calcium digestibility from Moringa oleifera, spinach and sweet potato leaves.

(Mean ± SD; N = 3 replicate experiments on triplicate samples.)

Sample1 Digested Calcium %(g solubilized Ca/100 g Ca in dried leaf)

M3 21.68 ± 2.65

M4 40.48 ± 3.42

M5 37.24 ± 1.66

S 1.63 ± 0.08

SP 3.01 ± 0.68

1

Samples: M3= Moringa sample from India, M4= Moringa sample from Karonga, Malawi, M5= Moringa sample from Lilongwe, Malawi, S= spinach, SP= sweet potato leaves.

2.2.3 Bioaccessibility of Calcium

The bioaccessibility of calcium from Moringa oleifera samples and the control samples, spinach and sweet potato leaves are provided in Table 2.4

Bioaccessability of calcium from the samples was expressed as percentage of the dry matter. Bioaccessible calcium was equivalent to dialyzable calcium. The bioaccessibility results

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Using Tukey’s Studentized Range Test at α=0.05, there was no significant difference in dialyzable calcium between Moringa oleifera samples from Malawi, and Moringa oleifera

sample from India.

Also, there was no significant difference in percentage dialyzable calcium between Moringa oleifera samples from India and Malawi (P < 0.05). The mean percentage dialyzable calcium was 6.82±1.80, 10.75±0.96 and 11.15 ±2.88 for Moringa oleifera samples from India (M3), from Malawi (M4) and the second sample from Malawi (M5) respectively. The mean percentage dialyzable calcium in spinach and sweet potato leaves were 0.55±0.37, and 3.08±2.05 respectively.

Table 2.4 also shows the percentage of the digestible calcium that was dialyzable. Several samples were deleted from this calculation because the calcium concentration in the dialysate was too low for accurate measurement. Despite the substantial variation in the data, the table

shows that about one third of the digestible calcium was dialyzable in the Moringa samples, which was about 50% greater than the dialyzability of the digestible calcium in spinach or

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Table 2.4: Calcium bioaccessibility from Moringa oleifera, spinach and sweet potato

leaves

Sample1 Dialyzable Calcium

(% of total)

Dialyzable Ca as % of Digestible Ca

M3 6.82 ± 1.80 32.2 ± 11.6

M4 10.75 ± 0.96 31.8 ± 10.4

M5 11.15 ± 2.88 33.4 ± 8.2

S 0.55 ± 0.37 21.1 ± 18

SP 3.08 ± 2.05 20.4 ± 4.1

1

Samples: M3= Moringa sample from India, M4= first Moringa sample from Karonga, Malawi, M5= second Moringa sample from Lilongwe, Malawi, S= spinach, SP= sweet

potato leaves.

2.5 Discussion

The in-vitro methods for assessing the bioaccessibility of essential minerals are widely used because they are simple, fast, and inexpensive. These methods have been used in different

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In their study, fresh vegetables were prepared by blanching in boiling deionized water for three minutes followed by homogenization in a food processor whereas seed and pod

samples were cooked and homogenized in an electric blender and all the samples were stored in polyethylene bottles at -20oC before analysis.

In our study however, Moringa oleifera leaf samples were air dried, pounded into powder and stored in polyethylene bags before being subjected to chemical analysis. The present

study assessed calcium content, digestibility and bioaccessibility in Moringa oleifera leaf powder from Malawi and India; spinach and sweet potato leaf powder were used as controls. Bioaccessibility of calcium was determined from dialyzed or absorbed calcium using dialysis

tubing. As observed from the results, there was a statistically significant difference in percentage calcium content from the Moringa oleifera samples from India and the two samples from Malawi. This may be due to differences in the growing conditions because some soils have higher amounts of calcium. The digested calcium in the two Moringa oleifera samples from Malawi was statistically similar and different from Moringa oleifera

sample from India. This may be due to similar growing conditions for the samples from Malawi and different growing conditions with samples from India, or due to genetic drift in

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However, dialyzed calcium from all the Moringa oleifera samples was significantly higher than the control samples of spinach and sweet potato leaves. A study by Kamchan et al.,

(2004) that used in-vitro methods to study calcium dialyzability from different vegetables found percentage dialyzable calcium in kale, celery and Chinese cabbage to be 38.9 ± 2.1,

36.2 ± 4.1 and 32.2 ± 4.6 respectively. This may be due to different chemical and biological composition of these plants that inhibit absorption of calcium such as phytate, oxalate, and dietary fiber.

Ezeike et al., (2011) were able to extract 0.28 mg of oxalate per gram of dry matter from

Moringa oleifera, and cited data to calculate that spinach contains 100 mg oxalate per gram of dry matter. Oxalic acid in fresh sweet potato leaves was 3 to 5 mg per gram fresh weight, or about 16 to 27 mg per gram of dry matter (Almazan, 1995). The results from the present

study are different from those found by Kamchan probably due to the use of different species of plants and different sample preparatory methods. In an in-vitro iron bioavailability study, Yung et al., (2002), found that boiling the fresh leaves and dried powder of Moringa oleifera

enhanced the in-vitro iron bioavailability by 3.5 and 3 times respectively. A similar preparatory method would enhance the calcium bioaccessibility in our study.

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The present study has found the percentage calcium content in powdered Moringa oleifera

leaves to be 1.67 ± 0.0014, 1.44 ± 0.0014 and 1.55 ± 0.00 for samples from India (M3), from

Karonga, Malawi (M4) and from Lilongwe, Malawi (M5) respectively. If we translate these results per 100-g edible sample, it yields 1.67 g (1670 mg), 1.44 g (1440 mg) and 1.55 g

(1550 mg) for M3, M4 and M5 respectively, averaging 1553 mg/100 g. These results are not very different from that of Price (1985) for the leaf powder which was 2003 mg per 100-g sample. The current calcium RDA for pregnant and lactating women are 1300 mg/day and

1000 mg/day for women aged 14-18 years and 19-50 years respectively (IOM, 2011).

A dietary intake study undertaken in Malawi, Africa, showed that calcium intakes from

women was lower than the recommended intakes with average intakes of 620 mg/day and 622 mg/day for non-lactating women and lactating women respectively.

Another nutrient intake study conducted in South Africa in three ethnic groups (black,

white and mixed ancestry subjects) found that mean dietary calcium was higher in white subjects than in black and mixed ancestry subjects; however, calcium intake was low in all

the groups with about half of the DRI of 1000 mg/day (Charlton et al., 2005). If the dietary intake of Moringa oleifera is increased, calcium intake could also be tremendously increased, especially in the developing countries like Malawi where these trees grow with little care.

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According to our study, only 64 g of dried leaf powder would supply the 1000 mg/day RDA. From this intake, 330 mg of calcium are estimated to be digestible, and 96 mg of calcium are

estimated to be bioaccessible, or available for absorption, based on the average of the

Moringa oleifera plant samples tested in our study.

2.6 Conclusion

The percentage dialyzed calcium is higher in Moringa oleifera leaves than other vegetable sources such as spinach and sweet potato leaves. Percentage dialyzed calcium of Moringa oleifera grown in Malawi is slightly higher than that grown in India. Because there is evidence from related research that percentage calcium dialyzability differs in different foods, especially vegetable foods due to presence of different inhibitory components, I

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2.7 References

Almazan AM (1995) Antinutritional factors in sweet potato greens. J Food Comp Anal 8: 363-368.

Dahot MU, and Memon AR (1985). Nutritive significance of oil extracted from Moringa oleifera seeds. J Pharm University Karachi 3(2):75-80. NUT

Ezeike CO, Aguzue OC,Thomas SA (2011) Effect of brewing time and temperature on the

release of manganese and oxalate from Lipton tea and Azadirachta indica(Neem),

Phyllanthus amarus and Moringa oleiferablended leaves. J Appl Sci Envir Manag 15:175-177.

Ferreira PMP, Farias DF, Oliveira JTA, Carvalho AFU (2008) Moringa oleifera: bioactive compounds and nutritional potential. Revista de Nutricao Campinas 21: 431-437.

Fuglie JL (2005). The Miracle Tree: a local solution to malnutrition? Church World Services in Senegal.

Kamchan A, Puwastien P, Sirichakwal PP and Kongkachuichai R, (2004)In vitro calcium bioavailability of vegetables legumes and seeds. J Food Comp Anal17:311-320.

Muluvi GM, Sprent JI, Soranzo N, Provan J, Odee D, Folkard G, Mcnicol JW, Powell W (1999) Amplified fragment length polymorphism (AFLP) analysis of genetic variation in

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Oduro I, Ellis WO, and Owusu D (2008) Nutritional potential of two leafy vegetables:

Moringa oleifera and Ipomea batatas leaves. Sci Res and Ess 3(2): 57-60.http://www.academicjournals.org/SRE

Price ML, (1985). The Moringa Tree. ECHO Technical Note. Educational Concerns for

Hunger Organization, N. Ft. Meyers, FL. http://echonet.org/repositories - 110:d:The Moringa

Tree cited 12/06.2011

Siddhuraju P, Becker K (2003) Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves. J Agric Food Chem 51:2144-2155. NUT

Trees for Life (2005)

Moringa Book.http://www.treesforlife.org/project/moringa/book/default.asp. Nut Gen

Yang RY, Tsou SCS, and Lee TC(2002)Effect of cooking on in vitro iron bioavailability of

various vegetables.p130-142. In TC Lee and CT Ho (eds.), Bioactive compounds in foods: effect of processing and storage. American Chemical Society, Washington, D.C.

Yang RY, Chang LC, Hsu JC ( 2006) Nutritional and Functional Properties of Moringa

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Appendix 1-SAS output data for percentage Calcium content in M3, M4 and M5

The SAS System

The ANOVA Procedure

Class Level Information

Class Levels Values

GROUP 3 1 2 3

Number of Observations Read 6

Number of Observations Used 6

The SAS System

The ANOVA Procedure

Dependent Variable: M345 M345

Source DF

Sum of Squares

Mean

Square F Value Pr > F

Model 2 0.053829 0.026915 20186 <.0001

Error 3 0.000004 1.33E-06

Corrected Total 5 0.053833

R-Square Coeff Var Root MSE M345 Mean

0.999926 0.074289 0.001155 1.554333

Source DF Anova SS

Mean

Square F Value Pr > F

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48 The SAS System

The ANOVA Procedure

Tukey's Studentized Range (HSD) Test for M345

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error

rate than REGWQ

Alpha 0.05

Error Degrees of Freedom 3

Error Mean Square 1.33E-06

Critical Value of Studentized Range 5.90959 Minimum Significant Difference 0.0048

Means with the same letter are not significantly different.

Tukey Grouping Mean N GROUP

A 1.671 2 1

B 1.553 2 3

C 1.439 2 2

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49 The SAS System

The ANOVA Procedure

Scheffe's Test for M345

Note: This test controls the type 1 experimentwise error rate

Alpha 0.05

Error Mean Square 1.33E-06

Critical Value of F 9.55209

Minimum Significant Difference 0.005

Scheffe Grouping Mean N GROUP

A 1.671 2 1

B 1.553 2 3

C 1.439 2 2

Note: A=M3, B=M4, C=M5

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Appendix 2: SAS output data for % Digested Calcium in M3, M4, M5.

The SAS System

The ANOVA Procedure

Class Levels Values

Sample 3 M3 M4 M5

The SAS System

The ANOVA Procedure

Source DF

Sum of

Squares Mean Square

F

Value Pr > F

Model 2 606.150749 303.0753745 42.37

0.000 3

Error 6 42.9212402 7.15354

R-Square CoeffVar Root MSE M345 Mean

0.933873 8.073 2.67461 33.13209

Source DF Anova SS Mean Square

F

Value Pr > F

Sample 2 606.150749 303.0753745 42.37

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51 The SAS System

The ANOVA Procedure

Note: This test controls the Type I experimentwise error

rate, but it generally has a higher Type II error rate than REGWQ

Alpha 0.05

Error Mean Square 7.15354

Critical Value of Studentized Range 4.33902

Tukey Grouping Mean N sample

A 40.481 3 M4

A 37.237 3 M5

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52 The SAS System

The ANOVA Procedure

Note: This test controls the Type I experimentwise error rate.

Alpha 0.05

Scheffe Grouping Mean N sample

A 40.481 3 M4

A 37.237 3 M5

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53 The SAS System

The ANOVA Procedure

Class Levels Values

Sample 3 M3 M4 M5

The SAS System

The ANOVA Procedure

Source DF

Sum of

Squares Mean Square

F

Value Pr > F

Model 2 34.0936764 17.0468382 4.26

0.070 6

Error 6 24.01240923 4.0020682

R-Square CoeffVar Root MSE M345 Mean

0.586749 20.92 2.000517 9.564889

Source DF Anova SS Mean Square

F

Value Pr > F

Sample 2 34.0936764 17.0468382 4.26

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54 The SAS System

The ANOVA Procedure

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ

Alpha 0.05

Critical Value of Studentized Range 4.339

Tukey Grouping Mean N sample

A 11.15 3 M5

A 10.72 3 M4

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55 The SAS System

The ANOVA Procedure

Note: This test controls the Type I experimentwise error rate.

Alpha 0.05

Scheffe Grouping Mean N sample

A 11.15 3 M5

A 10.72 3 M4

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Appendix 4- Raw Data for percentage Calcium content, %digested Calcium and %dialyzed Calcium

Samples

Initial inside

(ml) Initial outside (ml) Initial Ca Initial Ca(mg)

Final

Outside Final

A.A (ppm)

Ca A.A

(ppm) Volume(ml)

S 1-1 10 25 0.6 0.0067 0.1 35

S 1-2 10 25 0.5 0.0056 0.1 35

S.P 2-1 10 25 1.6 0.0178 0.1 35

S.P 2-2 10 25 1.3 0.0144 0.1 35

M 3-1 10 25 3.6 0.04 0.2 35

M 3-2 10 25 3.9 0.0428 0.2 35

M 4-1 10 25 11.1 0.1233 0.4 35

M 4-2 10 25 5.8 0.0639 0.3 35

M 5-1 10 25 4.9 0.0544 0.4 35

M 5-2 10 25 5.3 0.0583 0.4 35

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57 Final Dialyzed

Ca (mg)

Ca Dialyzed

(%) Ca Digested(%) Ca Dialyzed of Dry matter(%)

Average dialyzed(%)

0.0039 58.33 1.6852 0.9830

0.9830

0.0039 70.00 1.4043 0.9830

0.0039 21.88 4.1850 0.9155

0.9155

0.0039 26.92 3.4003 0.9155

0.0078 19.44 23.9091 4.6490

4.6490

0.0078 18.18 25.5695 4.6490

0.0156 12.61 85.7673 10.8175

9.4653

0.0117 18.26 44.4290 8.1131

0.0156 28.57 35.6079 10.1737

10.1737

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58 Samples Initial inside (ml)

Initial outside (ml)

Initial Ca A.A

(ppm) Initial Ca(mg) Final Inside Final

Ca A.A (ppm)

Volume(ml)

S 1-1 10 25 0.6 0.0067 0.8 4.9

S 1-2 10 25 0.5 0.0056 0.8 5.3

S.P 2-1 10 25 1.6 0.0178 3.1 4.1

S.P 2-2 10 25 1.3 0.0144 2.5 4.5

M 3-1 10 25 3.6 0.04 6.2 4

M 3-2 10 25 3.9 0.0428 6.5 4.3

M 4-1 10 25 11.1 0.1233 20.9 3.5

M 4-2 10 25 5.8 0.0639 11.1 4

M 5-1 10 25 4.9 0.0544 8.2 4

M 5-2 10 25 5.3 0.0583 8.1 4.5