EFFECT OF WATER BLANCHING AND DRYING (CABINET AND

PAN) ON NUTRIENT, PHYTOCHEMICAL AND DPPH RADICAL

SCAVENGING ACTIVITY OF MORINGA (Moringa oleifera) LEAVES
AND ITS SENSORY ATTRIBUTES

by

Nitesh Parajuli

Department of food technology

Dharan Multiple Campus

Institute of food technology

Tribhuvan University, Nepal

2018
 Effect of Water Blanching and Drying (Cabinet and Pan) on Nutrient,
Phytochemical, and DPPH Radical Scavenging activity of Moringa
(Moringa Oleifera) Leaves and its Sensory Attributes

A dissertation submitted to the Department of Food Technology, Dharan multiple

Campus, Tribhuvan University, in partial fulfillment of the requirements for the degree
of B. Tech in Food Technology

by

Nitesh Parajuli

Department of Food Technology

Dharan Multiple Campus

Institute of food technology

Tribhuvan University, Nepal

2018
 Tribhuvan University

Institute of Science and Technology

Department of Food Technology

Dharan Multiple Campus, Dharan

Approval Letter
This dissertation entitled Effect of Water Blanching and Drying (cabinet and pan) on
Nutrient, Phytochemical and DPPH Radical Scavenging Activity of Moringa (Moringa
Oleifera) Leaves and its Sensory Attributes by Nitesh Parajuli has been accepted as the
partial fulfillment of the requirement for the B.Tech. degree in Food Technology.

Dissertation Committee

1. Head of the Department

(Mr. Pashupati Mishra, Prof.)

2. External Examiner

(Mr. Pramod Koirala. Chief, RFTQC)

3. Supervisor

(Mr. Yadav K.C., Lecturer)

4. Internal Examiner

(Mr. Arjun Ghimire, Lecturer)

April, 2019

ii
 Acknowledgements
First of all, I wish to express my deepest gratitude and sincere appreciation to my respected
guide Mr. Yadav K.C, CCT, Dharan for his helpful guidance, constructive criticism,
constant encouragement and frequent inspiration throughout the dissertation period, which
enable me to complete this dissertation.

I would like to convey my sincere gratefulness to Mr. Basanta Rai, Assoc. Professor,
CCT, Dharan for providing necessary research facilities during the work and for their kind
support and valuable suggestions.

I would also like to acknowledge the entire library and laboratory staffs of Dharan
Multiple Campus, CCT, Dharan, especially Mr. Sachin Basnet and Mr. Ankit Katwal for
their cooperation and support during the work. I would also like to thank all my friends
(Ruby Rai, Anuradha Yadav and Shrijana Shrestha) for their help and co-operation.

Last but not the least; I would like to express my deepest reverence to my respected
parents for their kind support and encouragement throughout the course of this study.

Date of Submission

(Nitesh Parajuli)

iii
 Abstract
Moringa oleifera is an important multipurpose tropical tree under recognized for its
nutritional and medicinal properties. The present study was undertaken to evaluate the
effect of water blanching and two (cabinet drying and dry roasting) methods on
physiochemical composition, DPPH radical scavenging activity of Moringa oliefera
leaves. The fresh leaves were collected from Tamor river banks from Dhankuta district,
Nepal. Initially adequate time-temperature for water blanching was optimized to be 98oC
for 2 min and for cabinet drying and dry roasting temperature are 50-55oC and 120-130oC
respectively. Fresh, blanched and dried leaves were analyzed for their nutrient,
phytochemicals content, antioxidant activity and vitamin C.

Proximate composition of fresh Moringa oleifera leaves were analyzed that is moisture,
protein, crude fiber, fat and ash content and found to be 75.6%, 24.85%, 8.19%, 7.1%,
8.72% respectively on dry basis except moisture content. Crude extract of sample were
prepared using 80% methanol through maceration technique. Phytochemical screening of
the methanol extract of the plant showed the presence of total phenol content, tannin,
glycosides and steroid. total phenol, flavonoid, tannin content, DPPH radical scavenging
activity assay, vitamin C and chlorophyll of fresh leaves were found to be 146.0 mg/g,
526.7 mg/g, 12.81 mg/g, 82.98 mg/g, 155.67 mg/100 g and 23.27 mg/g respectively. Thus,
results showed that drying, pan drying and blanching decreased the level of phytochemical
content and antioxidant activity (p<0.05) as compared to fresh leaves. Pan drying had a
significant effect on phytochemicals content, vitamin C, chlorophyll and antioxidant
activity of Moringa oleifera leaves. Fresh leaves are superior than that of others in terms of
phytochemical and blanching and cabinet drying are superior in sensory analysis which is
suitable for fortification of foods. This study reveals that fresh and blanched leaves are
more suitable as a dietary antioxidant than dry leaves. This plant can contribute
significantly to the daily recommended allowance needed for many vitamins and minerals
as well as serve as rich sources of phytochemicals and antioxidant.

iv
 Contents
List of Tables ..................................................................................................................... ix

Lists of Figure .................................................................................................................... x

List of plates ...................................................................................................................... xi

1. Introduction ................................................................................................................ 1-4

1.1 General introduction ............................................................................................. 1

1.2 Statement of the problem ...................................................................................... 2

1.3 Objectives ............................................................................................................. 3

1.3.1 General objective ...................................................................................... 3

1.3.2 Specific objectives .................................................................................... 3

1.4 Significance of the study ........................................................................................ 3

1.5 Limitation of the study .......................................................................................... 4

2. Literature review ...................................................................................................... 5-35

2.1 Historical background, distribution and varieties ................................................. 5

2.1.1 Origin and distribution ............................................................................. 5

2.1.2 Classification and morphology ................................................................. 5

2.1.3 Ecology and physiology ........................................................................... 6

2.2 Processing of moringa Leaves .............................................................................. 8

2.2.1 Blanching .................................................................................................. 8

2.2.2 Drying ..................................................................................................... 13

2.3 Chlorophylls ........................................................................................................ 16

2.4 Vitamin C (L-ascorbic acid) ............................................................................... 18

2.4.1 Physiochemical and biochemical functions of vitamin C ...................... 19

2.4.2 Effect of deficiency of vitamin C in humans.......................................... 20

2.4.3 Side effect from over dosage .................................................................. 20

2.5 Phytochemicals present in moringa leaves ......................................................... 20

v
 2.5.1 Phytochemical definition ........................................................................ 20

2.5.2 Classes of phytochemicals ...................................................................... 20

2.5.3 Phenolics / Polyphenols .......................................................................... 22

2.5.4 Flavonoids .............................................................................................. 24

2.5.5 Tannins ................................................................................................... 25

2.5.6 Phytochemical metabolism in human ..................................................... 27

2.6 Antioxidants ........................................................................................................ 27

2.6.1 Necessity of use of antioxidants ............................................................. 28

2.6.2 Mechanism of action .............................................................................. 28

2.6.3 Types of antioxidant ............................................................................... 29

2.6.4 Oxidative stress ...................................................................................... 33

2.6.5 Mechanism of action of antioxidants ..................................................... 33

2.6.6 Levels of antioxidant action .................................................................... 34

3. Materials and methods ........................................................................................... 36-47

3.1 Raw materials...................................................................................................... 36

3.2 Equipment and chemicals ................................................................................... 36

3.4 Collection and preparation of sample ................................................................. 38

3.5 Preparation of plant extracts ............................................................................... 40

3.6 Phytochemical qualitative analysis ..................................................................... 40

3.7 Quantitative analysis ........................................................................................... 42

3.7.1 Determination of moisture content ......................................................... 42

3.7.2 Determination of crude protein .............................................................. 43

3.7.3 Determination of crude fat...................................................................... 43

3.7.4 Determination of ash content.................................................................. 43

3.7.5 Determination of crude fiber .................................................................. 43

3.7.6 Determination of total phenol ................................................................. 43

vi
 3.7.7 Determination of flavonoids ................................................................... 44

3.7.8 Determination of tannins ........................................................................ 44

3.7.9 Determination of chlorophyll ................................................................. 44

3.7.10 Determination of Ascorbic acid (vitamin C) ........................................ 45

3.7.11 Determination of blanching time .......................................................... 45

3.7.12 Determination of DPPH radical scavenging activity............................ 45

3.8 Sensory analysis .................................................................................................. 45

3.8 Optimization of moringa Powder........................................................................ 46

3.9 Statistical analysis ............................................................................................... 47

4. Results and discussion ............................................................................................ 48-66

4.1 Physicochemical properties of moringa Leaves ................................................. 48

4.2 Optimization of blanching time .......................................................................... 50

4.3 Qualitative phytochemical screening of Moringa oleifera leaves ...................... 52

4.4.1 Effect of processing conditions on total phenol content ........................ 53

4.4.2 Effect of processing conditions on total flavonoid content .................... 55

4.4.3 Effect of processing conditions on tannin content ................................. 56

4.4.4 Effect of processing conditions on chlorophyll content ......................... 57

4.4.5 Effect of processing conditions on antioxidant activity ......................... 58

4.5 Sensory evaluation of Moringa oleifera ............................................................. 60

4.5.1 Color ....................................................................................................... 61

4.5.2 Flavor ...................................................................................................... 62

4.5.3 Taste ....................................................................................................... 63

4.5.4 After taste ............................................................................................... 64

4.5.5 Overall acceptability............................................................................... 65

4.6 Optimization study .............................................................................................. 66

vii
5. Conclusion and recommendations ............................................................................. 68

5.1 Conclusion .......................................................................................................... 68

5.2 Recommendations ............................................................................................... 68

6. Summary ...................................................................................................................... 70

References ................................................................................................................ 71-84

Appendices ............................................................................................................... 85-97

viii
 List of Tables

Table No List of Table Page No

2.1 Moringa oleifera requirement and ranges 6

2.2 Time-temperature relationship of blanching 9

2.3 Phytochemicals present in plants 22

3.1 List of chemicals used 35

3.2 Lists of equipment‟s used 36

3.3 Optimization of phytochemicals parameters 44

3.4 Optimization of sensory parameters 45

4.1 Physicochemical properties of fresh, blanched dried, cabinet dried 47

and dry roasted leaves

4.2 Blanching time-temperature optimization 49

4.3 Qualitative analysis for phytochemicals 50

4.4 Quantitative analysis for phytochemicals 51

4.5 Sensory evaluation 58

4.6 Optimized goals for sensory analysis 65

4.7 Optimized goals for phytochemical analysis 65

ix
 Lists of Figure

Figure No. List of figure Page No.

2.1 Movement of moisture during drying 13

2.2 Pathways for degradation of chlorophyll 17

2.3 Structure of flavonoids 24

2.4 Mechanism of action of antioxidants 28

2.5 Chemical structure of TBHQ 30

2.6 Chemical structure of BHA 30

2.7 Chemical structure of BHT 31

3.1 Flow diagram of methodology 38

4.1 Mean sensory score for color 59

4.2 Mean sensory score for flavor 60

4.3 Mean sensory score for taste 61

4.4 Mean sensory score for after-taste 62

4.5 Mean sensory score for overall acceptability 63

x
 List of plates

Plates No. List of Plates Page No.

P1 Fresh moringa leaves 94

P2 Extract preparation 94

P3 Sensory analysis 95

P4 Phytochemical analysis 95

xi
 List of abbreviations

Abbreviation Full form

AlCl3 Aluminium Chloride

BHT Butylated hydroxyl toluene

DPPH 2,2-Diphenyl 1,1 picrylhydrazyl

FeCl3 Ferric Chloride

GAE Gallic Acid Equivalent

HCl Hydrochloric Acid

H2SO4 Sulphuric acid

Na2CO3 Sodium Carbonate

NaHCO3 Sodium Biacarbonate

NaNO2 Sodium Nitrate

NaOH Sodium Hydroxide

TBHQ Tert-Butylhydroquinone

TFC Total Flavonoid Content

TPC Total Phenol Content

xii
 Part I

Introduction

1.1 General introduction

Medicinal herbal drugs play a key role in health especially in the developing countries like
Nepal, Ethiopia. Currently herbal medicines are used to treat common diseases such as
typhoid, pneumonia and malaria due to the fact that they are cheaper, easily available and
are believed to present lesser side effects. More than 80% of the population within
developing countries relies on the use of herbal medicines for their primary healthcare due
to their lower cost and time tested nature (Hussain et al., 2009). The medicinal values of
plants are therefore, attributed to pharmacologically active compounds that have no direct
impact on the plants main processes but research has proved these compounds to have
great medicinal values. These compounds, which are used by the plant to protect it against
predators, are called secondary metabolites or phytochemicals (Hailu, 2015).
Phytochemicals with different bioactivities in various vegetable crops are important
components of a healthy diet. Many of these compounds appear to be responsible for the
high antioxidant activity that is beneficial to human health. Currently, people pay more
attention to the nutrition and quality of the food they consume, thus increased levels of
these health-promoting substances in vegetables would be more desirable to meet
consumer demand (Chanli, 2012).

Moringa oleifera is the most widely distributed and naturalized species of a

monogeneric family; moringaceae (Nadkarni, 1976; Ramachandran et al., 1980). It is a
perennial plant that grows very fast with flowers and fruits appearing within 12months of
planting. They grow up to the height of 5-12 m and pods 30-120 cm long (Morton, 1991).
Moringa oleifera, native of the western and sub-Himalayan tracts, India, Pakistan, Asia
minor, Africa and Arabia (Somali et al., 1984) and is now distributed to the Philippines,
Cambodia, Central America, North and South America and the Caribbean Islands (Morton,
1991). In some parts of the world, Moringa oleifera is referred to as the “Drumstick tree or
the Horse radish tree” where in other, it is also known as the Kelor trees (Amwar and
Bhanger, 2003). It is called sitalchini, shaijan in Nepali. They are available in the mid-hills
and Terai of Nepal. Moringa is considered one of the most useful trees, as almost every
parts of the tree can be used for food and have other beneficial property. Oil extracted from

1
the seeds is used for working machinery, Cosmetics, cooking and soap (Singh and Prasad,
2013). The press cakes, which is left after the oil extraction is used as soil fertilizers. Pods
and leaves are used for eating by humans and animals, as they contains lots of vitamins,
proteins and good sources of natural antioxidants like ascorbic acid, flavonoids, phenolic
and carotenoids (Becker and Siddhraju, 2003; Dillard and German, 2000). The high
concentration of ascorbic acids, oestrogenic substance, iron calcium, phosphorous, coppers
vitamin A, B and C, α-tocopherol, riboflavin, nicotinic acid, folic acid, pyridoxine, β-
carotene, protein and in particular essential amino acids such as methionine, cysteine,
tryptophan and lysine, phytochemicals and antioxidant present in Moringa oleifera leaves
and pod make it virtually ideal dietary supplement (Makkar and Becker, 1996).

1.2 Statement of the problem

Studies made on Moringa oleifera leaves have shown that like higher plant leaves, it is rich
in protein. It contains about 27% protein on a dry weight basis. It is also rich in calcium,
potassium, vitamins A, B and C which are important constituents of human diet. Presence
of several nutrients like protein, carbohydrates, vitamins, minerals, lipids it has several
health benefits (Seshadri and Nambiar, 2003). Cooked leaf of Moringa oleifera contains
beta-carotene, folic acid, iron which protects against anemia. Various research have shown
that moringa leaves have helped in digestion, good eye vision, prevent from cancer,
reduces high cholesterol blood sugar, also acts as cardiac and circulatory stimulants,
possess antitumor, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antibacterial and
antifungal activities. It is also useful for the lactation mothers to increase woman‟s milk
production, also effective in treating malnutrition, protects cells from being damaged from
the free radicals as it contains enormous amount of phenolic, flavonoids, carotenoids
compounds which tend to exhibit potent antioxidant properties. It prevent from anemic,
fatigue weakness and tiredness. Eating moringa leaves on a regular basis prevents wrinkles
and rejuvenates the skin at the same time (Jed and Fahey, 2005).

As all leafy vegetables are perishable products. Likewise, Moringa oleifera leaves has
also short shelf-life due to high moisture content and no any steps have been put forward
for its preservation in stable form. Hence to promote the consumption of such important
plants, it needs to be preserved in a stable form compatible for consumption. On the other
hand, moringa leaves are found elsewhere, cheap compared to its pods, its

2
commercialization as stable products will be the best means to raise life standard of the
local people (Mugal and Haq, 2010).
1.3 Objectives

1.3.1 General objective

The general objective of my dissertation work is to study the effect of water blanching and
drying (cabinet and pan) on nutrient, phytochemical and DPPH radical scavenging activity
of Moringa oleifera leaves and its sensory attributes.

1.3.2 Specific objectives

i. To prepare 80% methanolic extract of the samples.

ii. To determine the nutritive value of fresh, blanched, cabinet dried and pan dried
leaves.
iii. To study the effect of blanching, cabinet drying and pan drying on phytochemical
composition of leaves.
iv. To evaluate the DPPH radical scavenging activity assay of the extracts of the
collected samples.
v. To evaluate the sensory analysis of samples.

1.4 Significance of the study

Moringa oleifera is rich sources of protein, minerals; vitamins compared to others leafy
vegetables. Moringa leaves are cheap and easy to consume. They contain wide variety of
biologically active, non-nutritive compounds known as phytochemicals which imparts
health benefits. In a country like Nepal with countless malnourished people, introduction
of Moringa oleifera of antioxidants along with medicinal values can uplift the standard of
local people. The present study is therefore a preliminary effort towards finding the effect
of processing methods on the bioactive compounds of Moringa oleifera. It has been long
pleused in traditional medicine and food supplements by native people. Exploration of
phytochemical properties of M. oleifera may help to support its traditional climb. High
population growth and increased resistance to antibiotics by bacteria has put high demand
on the use of natural medicinal plants. Since M. oleifera constituents a large group of
chemical with high potential to treat various diseases, phytochemical analysis is the best
way to bring forward M. oleifera as medicinally important plant.
3
1.5 Limitation of the study

 Single extraction technique was used to prepare extract solution.

 Identification of the phytochemicals was not carried out.

4
 Part II

Literature review

2.1 Historical background, distribution and varieties

2.1.1 Origin and distribution

Moringa oleifera is one of the most important medicinal plant and the most widely
cultivated species of moringaceae family, which is native to the sub Himalayan tracts of
India, Afghanistan, Pakistan, and Bangladesh. M. oleifera tree was used by Egyptian,
Greeks and ancient Romans. It is highly valued from ancient time because of its immense
medicinal properties (Chumak et al., 2008). M. oleifera is indigenous of Himalayas of
North-western India. This species is located in the sub-Himalayan tracts from the river
Chenad to eastwards Sarda and tract of Uttar Pradesh in India. Later this species has been
cultivated to others parts of the continent such as Malaysia, Philippines, Singapore, Sri
Lanka, Cuba and Burma. In Africa, the tree have been distributed in Nigeria, Egypt and
Sudan, also spread to central and south America, Peru to Mexico, Paraguay and others
cities in the world (Ramachandran et al., 1980).

2.1.2 Classification and morphology

Scientific Classification

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Brassicales

Family : Moringaceae

Genus : Moringa

Species : oleifera

There are thirteen species that are found in moringaceae. These are Moringa oleifera,
Moringa arborea, Moringa borziana, Moringa concanensis, Moringa drouhardii, Moringa

5
hildebrandtii, Moringa longituba, Moringa ovalifolia, Moringa peregrine, Moringa
pygmaea, Moringa rivae, Moringa ruspoliana, and Moringa stenopetala (Mughal and
Haq, 2010) Moringa oleifera is grown in many parts world including Nepal and is the most
studied species.

M. oleifera is a fast-growing, deciduous tree that can reach a height of 10–12 m (32–40
ft) and trunk diameter of 45 cm (1.5 ft).The bark has a whitish-grey color and is
surrounded by thick cork. Young shoots have purplish or greenish-white, hairy bark. The
tree has an open crown of drooping, fragile branches and the leaves build up feathery
foliage of tripinnate leaves (Leone et al., 2015). The flowers are fragrant and bisexual,
surrounded by five unequal, thinly veined, yellowish-white petals. The flowers are about
1.0-1.5 cm (1/2") long and 2.0 cm (3/4") broad. They grow on slender, hairy stalks in
spreading or drooping flower clusters which have a length of 10–25 cm. Flowering begins
within the first six months after planting. In seasonally cool regions, flowering only occurs
once a year between April and June (Olson and Carlquist, 2010). In more constant seasonal
temperatures and with constant rainfall, flowering can happen twice or even all year-round.
The fruit is a hanging, three-sided brown capsule of 20–45 cm size which holds dark
brown, globular seeds with a diameter around 1 cm. The seeds have three whitish papery
wings and are dispersed by wind and water. In cultivation, it is often cut back annually to
1–2 m (3–6 ft) and allowed to regrow so the pods and leaves remain within arm's reach
(Amaglo, 2010).
2.1.3 Ecology and physiology

Readily colonizes stream banks and savannah areas where the soils are well drained and
the water table remains fairly high all the year round. It is quite drought tolerant but yields
much less foliage where it is continuously under water stress. It is not harmed by frost, but
can be killed back to ground level by a freeze. It quickly sends out new growth from the
trunk when cut, or from the ground when frozen (Foidl et al., 2001).

It is grow between the altitudes 0-2000 m. Its mean annual temperature is 12.6 to 48oC
and means rainfall is at least 500 mm. It is adapted to a wide range of soil types but does
well in well drained clay or clay loam without prolonged water logging. It prefers a neutral
to slightly acidic soil reaction, but it has recently been introduced with success in Pacific at
where the pH is as high as 8.5 (Odee, 1998; Olson and Carlquist, 2010) which is shown in
Table 2.1..

6
Table 2.1 Moringa oleifera requirements and ranges

Requirement/range

Climate Grows best in tropical or subtropical

Altitude 0 – 2000 m

250 – 3000 mm
Rainfall
Irrigation needed for leaf production if rainfall < 800 mm

Soil type Loamy, sandy, or sandy-loam

Soil pH 5–9

Source: (Olson and Carlquist, 2001)

2.1.4 Moringa in Nepal

Moringa tree is already available around mid-hill and Terai area of Nepal but is not well
known. This plant can be grown together with other crops and can be a cost effective
nutritional supplement in the family food intake. People in Eastern Nepal use its pod for
vegetable and pickle making. In response to the environmental degradation, climatic
change, and the needs for nutritional supplements at the community level, Moringa
oleifera farming has and can become a part of rural development activities. Moringa is
grown traditionally as backyard trees or hedges. The increased awareness of the multiple
uses of moringa leaves for both domestic and industrial purposes is leading to an increased
demand for it. The moringa crop is suitable for more intensive production. In grows well in
most subsistent farms as an intercrop in association with other crops, producing a
significant amount of leaves (Pariyar, 2008). Due to moringa‟s many uses, it can provide
benefits to Nepal‟s agriculture industry. Nepal is home to millions of livestock with high
crop consumption, straining the resources available for people. One study found that
adding both fresh and dried moringa leaves to cattle feed significantly increases both milk
production and weight gain. As well, many of Nepal‟s central cereal crops are struggling
with decreasing rainfall due to climate change. Moringa is a highly drought tolerant

7
alternative, as it is able to survive with minimal rainfall and does not require irrigation
(Pokhrel et al., 2016).

The Terai, Siwalik, and Middle Mountain regions are the best-suited regions of Nepal
for moringa cultivation due to their elevation and climate conditions. Nepal found that
moringa growing naturally or planted randomly in about 40 districts including mid-hill
district of Dhankuta, Sindhuli, Ramechhap, Kavre, Kaski and Arghakhachi and adjoining
districts which have similar agro-climatic situations in Nepal. Terai and Mid-hills of Nepal
below 1500 m is suitable domains for cultivation of moringa in Nepal. Lower the altitude
betters the productivity. Rough estimates suggest that about 50% low lying and dry area of
Nepal is suitable for moringa cultivation (Khatri, 2014). In Nepal it has successfully grown
in home garden across the tropical region, foot hills, churia, and some part of mid hills.
Despite its multipurpose uses and potentiality for cultivation, this plant has not received
much attention for cultivation and conservation in Nepal (Pokhrel et al., 2016).

2.2 Processing of moringa Leaves

2.2.1 Blanching

The special heat treatment in order to inactivate the enzymes is known as blanching.
Blanching is not in discriminate heating. Too little is ineffective and too much is damages
the vegetables by excessive cooking, especially when the fresh character of the vegetable is
subsequently to be preserved by processing. This heat treatment is applied according to and
depends upon the specificity of vegetables, the objectives that are followed and the
subsequent processing / preservation methods (Margaret et al., 2017).

Two of the more heat resistant enzymes important in vegetables are catalase and
peroxidase. If these are destroyed then the other significant enzymes in the vegetables also
will have been inactivated. The heat treatment to destroy catalase and peroxidase in
different vegetables are known and sensitive chemical tests have been developed to detect
the amount of enzyme that might survive a blanching treatment. Because various types of
vegetables differ in size, shape, heat conductivity, and the natural levels of their enzymes,
blanching treatment have to be established on an experimental basis. As with sterilization
of foods in cans, the larger the food items, the longer it takes for heat to reach the center.
Small vegetables must be subsequently blanched in boiling water in a minute or two; large
vegetables may require several minutes (Mishra, 2007).
8
 Blanching is a unit operation is a short time heating in water at a temperature of 100⁰C
or below. Water blanching may be performed in double bottomed kettles, in special baths
in conveyor belts or in modern continuous blanching equipment. In order to reduce losses
of hydro soluble substances (mineral salts, vitamins, sugars, etc.) occurring during water
blanching, several methods have been developed (Jones, 1996).

i. Temperature setting at 85-95⁰C instead of 100⁰C.

ii. Blanching time has to be just sufficient to inactivate enzymes catalase and
peroxidase.

Steam heat treatment can also be applied instead of water blanching as a preliminary
step before freezing or drying, as long as the preservation method is only used for enzyme
inactivation and not to modify consistency.

According to (Odedeji et al., 2014) there was increase in the protein value after
blanching. This might be due to elimination of anti-nutritional factor tannin by the heat of
blanching, tannin is found to precipitate protein in the raw sample making it unavailable
for utilization (Oboh, 2006). The ash content in the blanched sample was found more than
fresh sample. Higher ash content is an index of mineral constituents. Similarly, increase in
carbohydrate content was found in the blanched sample, this might be due to the
destruction of the anti-nutritional factors binding the nutrients in the raw sample. But,
vitamin C was found to decrease after blanching the sample. This might be due to the heat
liability of the sample. An illustration of the blanching parameters is shown in the Table
2.2.

Table 2.2 Time temperature relationship of blanching of some vegetables

Vegetables Temperature, (°C) Time, min

Peas 85-90 2-7

Green Beans 90-95 2-5

Cauliflower Boiling 2

Carrots 90 3-5

Pepper 90 3

9
2.2.1.1 Effect on foods by blanching

The heat received by the food during blanching inevitably causes some changes to sensory
and nutritional qualities. In general, the time- temperature combination used for blanching
is a compromise which ensures adequate inactivation but prevents excessive softening and
loss of flavor in the food (Fellows, 2000). Small vegetables may be adequately blanched in
boiling water in a minute or two but large vegetables may require several minutes (Mishra,
2007). Some of the effects on foods are discussed below:

2.2.1.1.1 Nutrients

Some minerals, water-soluble vitamins and other water-soluble components are lost during
blanching. Losses of vitamins are mostly due to leaching, thermal destruction and, to a
lesser extent, oxidation (Fellows, 2000; Horner et al., 1942). The extent of vitamin loss
depends upon on a number of factors including:

 The maturity of food and variety.

 Methods used in preparation of the food, particularly the extent of cutting, slicing
or dicing.
 The surface-area-to volume ratio of the pieces of food.
 Method of blanching.
 Time and temperature of blanching (lower vitamin losses at higher temperature for
shorter times).
 Method of cooling

2.2.1.1.2 Color and flavor

Blanching brightens the color of some foods by removing air and dust on the surface and
thus altering the wave length of reflected light. The green color of chlorophyll is protected
by using alkaline blanching, although the increase in pH may increase losses of ascorbic
acid. Blanching water is often added with sodium carbonate to neutralize the natural
acidity of the products. When, correctly blanched, most foods have no significant changes
to flavor or aroma, but under blanching can lead to the development of off- flavors during
storage of dried or frozen foods (Fellows, 2000; Garrote et al., 1984). Green tender peas
when blanched with the use of blanching aids, 0.125% MgO and 0.1% NaOHCO3 retained
maximum percentage of Chlorophyll (Kumar, 1991).

10
2.2.1.1.3 Texture

One of the purposes of blanching is to soften the texture of vegetables to facilitate filling
into containers prior to canning. Calcium chloride (1-2%) is added to the blanched water to
form insoluble calcium pectate complexes and thus to maintain firmness in the tissue
(Fellows, 2000).

Blanching is essential where fruits and vegetables are to be frozen or dried because
drying or freezing operations only slow down enzymatic action but do not completely stop
it. If blanching is not done prior freezing or drying then the frozen or dried product, which
is often held in frozen or dried state for many months, will slowly develop off flavors and
off colors and also other kinds of enzymatic spoilage might result. Under blanching may
cause more damage to food than the absence of blanching does. Heat, which is sufficient to
disrupt tissue but not to inactivate enzymes, causes the mixing of enzymes and substrates.
In addition, only some enzymes may be destroyed which causes increased activity of other
enzymes and thus accelerates deterioration (Kharel and Hashinaga, 2004; Saranaya et al.,
2017).

2.2.1.2 Methods of blanching

During blanching a raw food material is immersed in hot water or exposed to live steam.
Water temperature must be well controlled at desired level. The blanching operations
varies according to the maturity and type of vegetable used. In practice, water immersion
blanching and steam blanching are two general methods of blanching. Less frequently,
microwave blanching can be used (Kharel and Hashinaga, 2004).

2.2.1.2.1 Water blanching

It involves passing the food at a controlled rate through a perforated drum rotating in a tank
of water which is thermostatically controlled to the blanching temperature (70-100⁰C). In a
small plant, food to be blanched is passed on the wire of a perforated basket, which is first
dipped in hot water for a short period of time (2-5 min) and then in cold water. Hard water
toughens tissues and destroys the natural texture of foods. A disadvantage of immersion
blanching is that water soluble nutrient will pass into the blanching water, but an important
advantage is that undesirable oxidation can be easily controlled by appropriate additions to
the blanching bath (Bourne et al., 1979; Steinbuch, 1976).

11
2.2.1.2.2 Steam blanching

It uses saturated steam at atmospheric or at low pressure (150 KN/m2). The food is
conveyed through the steam chamber on a mesh belt or by the means of a helical screw, the
residence time being controlled by the conveyer speed. Typical equipment is 15 m long, 1
to 1.5 m wide and up to 2 m high. In convectional blanching there is often poor uniformity
of heating in the multiple layers of food. The time temperature combination required,
ensuring enzyme inactivation at the center of the bed results in the overheating of food at
the edges and this results in losses in texture and other sensory characteristics of the food,
individual quick blanching which involves blanching in two stages, overcomes this
problem (Corcuera et al., 2010).

In the first stage, food is heated in a single layer at a required temperature, and in the
second stage, a deep bed of food is held for sufficient time to allow complete enzymes
inactivation. This reduces the steaming time from a conventional 3 min to about 75 s (25 s
for heating and 50 s for holding). The blanched product is discharged through an outlet into
a cooler (Adam, 1981).

2.2.1.2.3 Microwave blanching

Microwave blanching has been applied to fruits and vegetables packaged in film bags and
would appear to offer some advantages such microbiological cleanliness and low losses of
nutrients. Blanching with microwave energy in order to apply heat at the center of large
items before the surface are overcooked, is receiving interest in application but is not yet
used commercially on a large scale due to its high cost (Ramesh et al., 2002). For drying,
the vegetables are conveyed directly from steaming equipment to drying installation
without cooling. Vegetable steaming is carried out in continuous installation with conveyor
belts made from metallic sieves. Cooling of vegetables after water blanching or steaming is
performed in order to avoid excessive softening of tissues and has to follow immediately
after such operations; one exception is the case of vegetables for drying which can be
transferred directly to drying equipment without cooling (Carroad et al., 1980). Natural
cooling is not recommended because is too long and generates significant losses in vitamin
C content. Cooling is pre-cooled air (from special installations) is sometimes used for
vegetables that will be frozen. Cooling in water can be achieved by sprays or immersion; in
any case the vegetables have to reach a temperature value under 37oC as soon as possible.

12
Too long a cooling time generates supplementary losses in valuable hydro soluble
substances; in order to avoid this, the temperature of cooling water has to be as low as
possible (Adam, 1981).

2.2.2 Drying

Drying is possibly the oldest method of food preservation. It has been known that the
various fruits and vegetables can be enjoyed out of season if the freshly harvested material
is preserved by drying (Shrestha, 2001).

2.2.2.1 Definition

From the engineering point of view, drying is the unit operation in which nearly all the
moisture in the food is removed by evaporation or sublimation as a result of application of
heat at controlled condition (Dinstel, 2017). This definition doesn„t include alternate
methods of moisture removal for example, filtration, membrane separation, centrifugation,
distillation, etc (MacCarthy, 1986).

2.2.2.2 Mechanism of drying

When hot air is blown over a wet food, heat is transferred to the surface, and latent heat of
vaporization causes water to evaporate. Water vapor diffuses through a boundary film of
air and is carried away by the moving air. This creates a region of lower water vapor
pressure at the surface of the food, and a water vapor surface gradient is established from
the moist interior of the food to the dry air. This gradient provides the driving force for
water removal from the food. Water moves to the surface by the following mechanisms:

 Liquid movement by capillary forces.

 Diffusion of the liquid which is adsorbed in layers at the surface of solid
components of the food.
 Diffusion of the liquids, caused by difference in the concentration of solutes in
different region of the food.
 Water vapor diffusion in air spaces within the food caused by vapor pressure
gradients.

Movement of moisture during drying is illustrated in Fig. 2.1

13
 Fig. 2.1 Movement of moisture during drying Kharel and Hashinaga (2004)

In foods, water is present as free water and bound water. Free or unbound water is
present within the pores and spaces between plant cells. It has the same characteristics as
pure water. It exerts the same vapor pressure and same latent heat of vaporization as pure
water. It is easily removed during drying. Bound water is held on the surfaces of solid
compounds, such as the cellulose, hemicelluloses, in the cell wall by molecular interactions
between water and the solid. It exerts a vapor pressure less than that of pure water, it does
not evaporate easily, and the latent heat of vaporization is greater than that of the pure
water at a given temperature. It requires more heat to release and therefore bound water is
not usually removed during drying (Heldman and Hartel, 1997)

During drying of a wet solid in a heated air, the air supplies the necessary sensible and
latent heat of evaporation of the moisture and also act as a carrier gas for the removal of
the water vapor formed from the vicinity of evaporating surface (Kharel and Hashinaga,
2004).

Many authors have worked on drying as a means of preserving agricultural products

including vegetables and fruits for storage purposes (Fohr and Arnaud, 1994).There are
many factors affecting the drying rate of the agricultural products. Among these factors,
we have air temperature, the relative humidity and the air velocity and initial moisture
content of the product. From literatures, the best range of temperature for drying fruits and
vegetables is between 55⁰to75⁰C. This falls within the range of the temperature obtained
from the solar dryer used to carry out this experiment (Awogbemi and Ogunleye, 2009).

2.2.2.3 Different methods of drying

The main methods of drying vegetables are as follows:

14
2.2.2.3.1 Sun drying

Sun drying is a natural method of drying making use of exposure to the sun and requires
time and some effort by the man to spread and collect the produce (Hall, 1970). Sun drying
has little or no operating cost, but is space consuming and has poor control over the drying
rates, which might enhance mold growth. Sun drying temperature varies with the variations
in the geographical regions (Williams et al., 1986). In tropics, a produce spread on matting
in a layer not thicker than about 5 cm receives a drying temperature of about 36⁰C on an
average (Hall, 1970). In such slow drying process, vitamin C content of the vegetable
tissue will be poorly retained (Desrosier and Desrosier, 1987).

2.2.2.3.2 Solar Drying

Solar drying refers to methods of using sun‟s energy for drying but excludes open air sun
drying. Solar drying is more effective than sun drying and has lower operative cost than
mechanized dryers. A simple principle involved in solar dryer is that, air is drawn through
the dryer by natural convection. The air is heated as it passes through the collector and then
partially cooled as it picks up moisture from the produce. The produce is heated both by
the air and directly by the sun (Teng and Chen, 1999).

2.2.2.3.3 Cabinet drying

Cabinet drier consist of an insulated cabinet fitted with shallow mesh or perforated trays,
each of which carries a thin layer of food. Hot air is circulated through the cabinet tray. A
system of duct and baffles is used to direct air, over and/ or through each tray, to promote
uniform air distribution. Cabinet drying are usually used for small scale operations
comparatively inexpensive, low maintenance cost, commonly used to dry fruit and
vegetable pieces (Fellows, 2000).

2.2.2.3.4 Dry roasting

It is the process by which the heat is applied to the food stuffs without the use of oil or
water as carrier. Unlike other dry heat methods, dry roasting is used with food stuffs such
as nuts and seeds, sometimes for the production of pan roasted tea leaves. Kamaricha,

Lung ching Dragon is some pan-dried tea. Dry roasting is stirred as they are roasted to
ensure even heating (Nicoli et al., 1999).

15
 It can be done in a frying pan, wok pan or in a specialized roaster. Dry roasting changes
the chemistry of protein, changing their flavor and enhances the scent and taste. In tea
leaves, roasting apparently make the leaf firm giving the tea a clear, yellow-green faint,
reddish taint, the taste is light and understated. Commonly dry roasted food include peanut
butter which is made from peanuts that have been roasted, tea, coffee beans, cocoa beans,
spices have been dry roasted either immediately after plucking or after fermentation
(Anonymous, 2008).
2.3 Chlorophylls
Chlorophylls, the pigments responsible for the characteristic green color of fruits and
vegetables, are highly susceptible to degradation during processing, resulting in color
changes in food (Schwartz and Von Elbe, 1983). The major chlorophylls in plants include
chlorophyll a and chlorophyll b, which occur in the approximate ratio of 3:1 (VonElbe and
Schwartz, 1996). Chlorophyll a has a methyl group at the C-3 carbon, while a formyl group
is bonded to the same carbon atom in chlorophyll b. In addition to structural differences
between chlorophyll a and b, their thermal stabilities are also different. Chlorophyll a was
reported to be thermally less stable than chlorophyll b (Canjura et al., 1991).

Chlorophyll retention has been used as a measure of quality in green vegetables. It is

well-known that the excessive heating of food products causes considerable losses in the
organoleptic quality of food. Chlorophyllase also hydrolyzes the phytol chain of
pheophytins giving rise to pheophorbides (Heaton and Marangoni, 1996).It is reported that
this enzyme acts at ambient temperature only in the presence of high concentrations of
organic solvents and only at temperatures between 65°C and 75°C in aqueous medium. On
the other hand, chlorophyllase losses its activity when heated to 100°C. Since the optimum
temperature for chlorophyllase activity in vegetables ranges between 60°C and 82.2°C,
some green vegetables show considerable formation of chlorophyllides and some
pheophorbides only at low-temperature(60-70°C)blanching treatment. Blanching
inactivates chlorophyllase and enzymes responsible for senescence and rapid loss of green
color. However, chlorophyll degradation is initiated by damaged tissue during blanching
and other processing steps (Tijekens et al., 2001).

Chlorophylls are susceptible to many chemical or enzymatic degradation reactions. The

simultaneous actions of enzymes, weak acids, oxygen, light and heat can lead to the
formation of a large number of degradation products. Major chemical degradation routes

16
are associated with pheophytinization, epimerization, and pyrolysis, and also with
hydroxylation, oxidation or photo-oxidation, if light is implicated. There is general
agreement that the main cause of green vegetable discoloration during processing is the
conversion of chlorophylls to pheophytins by the influence of pH. The green color of
vegetables turns to an olive green when heated or placed in acidic conditions (Gunawan
and Barringer, 2000). During this reaction, hydrogen ions can transform the chlorophylls to
their corresponding pheophytins by substitution of the magnesium ion in the porphyrin
ring. The conversion of chlorophyll to pheophytin and pheophorbide results in a change
from bright green to dull olive-green or olive-yellow, which is ultimately perceived by the
consumer as a loss of quality (Mangos and Becker, 1997). All of these reactions are
summarized in the Fig. 2.2.

Chlorophyll
(Green)

Mg+2 Strong acid

Acid Mg+2 phytol

Chlorophyllase
phytol

Pheophytins Acid Pheophorbides Acid Chlorophyllides

(Olive green) (Brown) (Bright green)

phytol Mg+2
O2
O2
O2 Acid

Chlorines, purpurins
(colorless product)

Fig. 2.2 Pathways for degradation of chlorophyll

A number of attempts have been made to preserve chlorophylls during heat processing
through the application of pH control (Heaton and Marangoni, 1996), high-temperature
short time processing or a combination of high temperature short time processing with pH
adjustments. Other improvements in color have involved the production of the more heat-
stable chlorophyllides. Alkalizing agents in blanch and brine solutions, such as sodium

17
bicarbonate, hexametaphosphate, disodium glutamate, sodium hydroxide, and magnesium
hydroxide, have been used to raise the pH of green vegetables and therefore, retain
chlorophyll after processing (Chen and Chen, 1993).

Since chlorophyll stability is known to be affected by pH and color is one of the most
important quality attributes of vegetable products, numerous studies have been conducted
to investigate the color changes or degradation of chlorophylls during heating and it is
reported that the chlorophyll degradation follows a first-order reaction kinetic model
(Canjura et al., 1991; Gunawan and Barringer, 2000). Chlorophylls have great importance
in food technology as well as other disciplines. In food technology, chlorophyll is related
to esthetic quality of fruits and vegetables. It is also extensively used as a parameter in
studying maturation and ripening of fruits and vegetables. In aquatic ecology, chlorophyll
determination is used for bio-monitoring (Rai, 2006). In Soviet Union, chlorophyll
extracted from the plant Stinging Nettle has been tried as a source of green pigment for use
in confectionary (Mishra, 2007).

2.4 Vitamin C (L-ascorbic acid)

L-ascorbic acid, which is also known as vitamin C, is an important naturally occurring

nutrient essential for human nutrition (Chauhan et al., 1998). Vitamin C is a white
crystalline compound with sour taste but no smell. Identification of its formula (C6H8O6)
was presented by group headed Professor Haworth in 1933 and it was the same group that
proposed the first synthetic method for its molecular weight is 176, and melting point is
190oC (Mottram, 1974). It is a derivative of glucose called Hexose, chemically it is 7-
threo-2,4,6, pentoxy hexen 2 carboxyllic acid lactone (Jain, 1996).

Ascorbic acid has four isomers, L-ascorbic acid, D-ascorbic acid, L-arabo ascorbic and
D-iso-ascorbic acid. Among the four isomers, the D-forms have no biological activity and
used as food additives. The presence of enediol group on ascorbic acid imparts acidic and
reducing properties. It behaves as mono basic and can give salt when reacted with alkalies.
Only l-ascorbic acid has important vitamin activity (Deman, 1976). The unusual properties
in the absence of carboxylic group, it has strong reducing properties and has one unusually
stable 1, 4-lactone ring (Hvoself, 1982).

18
 Ascorbic acid is highly soluble in water (30 g/100 ml), slightly soluble in alcohol and
insoluble in chloroform, ether and benzene (Jain, 1996; Mottram, 1974). It is very stable
when dry, moderately stable when dry, moderately stable in acid solution and unstable in
alkali. It is rapidly lost due to oxidation by exposure to the air, the oxidation is speeded up
by the heat, light, alkali oxidative enzymes and traces of copper and iron (Rajalakshmi,
1990). The ascorbic acid is precipitated by lead ion at pH 7.6 (Dasgupta, 1977).

It prevents diseases like scurvy and also tends to control to some extent many infectious
diseases, both viral and bacterial. It is also important for the healing of wounds, burns and
broken bones as it is required for the synthesis of all connective tissues (Heimann, 1980).
Diets rich in fresh fruits and vegetables are also protective against chronic, degenerative
diseases (Cox et al., 2000). Leafy vegetables have been known to be very vulnerable to
ascorbic acid loss. The vitamin C in fruits is readily available but the content in vegetable
might not be readily available due to various processing methods it undergoes such as
blanching, squeeze-washing, boiling and sun drying. It is the most easily destroyed of all
the vitamins. It is oxidized by oxidases contained within the cells of vegetables, which are
set free on cutting, chopping or crushing. A great percentage of it is lost to the water used
for washing and boiling vegetables because it is a water soluble vitamin. In home cooking
has been known to have quite a significant effect on the ultimate nutrient delivery to the
consumer particularly that of the labile, water-soluble ascorbic acid (Szeto et al., 2002).

2.4.1 Physiochemical and biochemical functions of vitamin C

The principle function of ascorbic acid is the formation of collagenous intercellular

substances. Ascorbic acid helps to reduce the ferric ion to ferrous state in the intestine and
thus helps in the absorption of iron. It is involved in the synthesis of cortical hormones and
metabolism of tyrosine (Swaminathan, 1991). A high dose of ascorbic acid depresses
alimentary hyper-cholesterol anemia and protects from the disease and resulting from
atherosclerosis. However, the effect on lipid oxidation as a function of ascorbic acid is a
controversial one (Ginter, 1974). Large dietary intake of ascorbic acid reduces the toxicity
of certain metals by increasing their urinary excretion as ascorbic acid complex. Ascorbic
acid is concerned with the formation of red blood corpuscles. A hemolytic state is
developed in the scurvy and is responsible for anemia. In the old age deficiency of ascorbic
acid is frequently observed, hence it is also regarded as anti-ageing agent (Jain, 1996).

19
2.4.2 Effect of deficiency of vitamin C in humans

The deficiency of vitamin C in human results in the defective formation of the intercellular
cement substances, Fleeting joint pains, irritability, retardation of the growth in infants or
child, anemia, poor wound healing and increased susceptibility to infection are among the
signs of deficiency (Swaminathan, 1991).

2.4.3 Side effect from over dosage

The reported side effects based on biochemical theory are gastro-intestinal disturbance,
increase peristalsis, abdominal colic, gastro-enteritis and anal irritation, looseness of
bowels, occasional diarrhea, abnormal uric acid metabolism, and production of gout, stone
formation, bone demineralization and collagen catabolism, calcium desorption, allergic
symptoms, haemolytic crisis, and human infertility. These effects were observed taking
daily dose in the range between 200 mg to 300 mg (Wilson et al., 1971).
2.5 Phytochemicals present in moringa leaves

2.5.1 Phytochemical definition

Phytochemicals are non-nutritive bioactive chemical compounds found naturally in plants

that protect against diseases. The active ingredients are the main effective compounds of
medicinal plants, the presence and quality vary from one plant to the other. A number of
phytochemical are known, some of which include: alkaloids, saponins, flavonoids, tannins,
glycosides, anthraquinones, steroids and terpenoids. They do not only protect the plants but
have enormous physiological activities in humans and animals. These include cancer
prevention, antibacterial, antifungal, antioxidative, hormonal action, enzyme stimulation
and many more (Doss and Anand, 2012).Many phytochemicals possess antioxidant activity
and reduces the risk of different diseases by serving structural and functional role as well
as an electrolyte (Abidemi, 2013).

2.5.2 Classes of phytochemicals

Phytochemicals are not essential nutrients and are not required by the human body for
sustaining life, but have important properties to prevent or to fight some common diseases.

Many of these benefits suggest a possible role for phytochemicals in the prevention and
treatment of disease, Because of this property; many researchers have been performed to

20
reveal the beneficial health effects of phytochemicals. The purpose of the present review is
to provide an overview of the extremely diverse phytochemicals presents in medicinal
plants. While phytochemicals are classified by function, an individual compound may have
more than one biological function serving as both an antioxidant and antibacterial agent
(Saxena et al., 2014). Bioactive and disease preventing phytochemicals present in plant are
shown in Table 2.3

Table 2.3 Phytochemicals present in plant

Phytochemical Phytochemicals Sources Potential

classes nutritionalbenefits

Carotenoids β-carotene,& Tomato, pumpkin, - Act as antioxidant

carotene, Carrot, watermelon.
- Reduce level of cancer
Guava, dark yellow
lutein, lycopene
pink and red - Producing enzymes
coloured vegetative
- Inhibit spread of cancer
fruits

Polyphenols Tannins Fruits, Legumes, -Exhibit anti-microbial and

Green vegetable, antioxidant
black
activities
Tea
- Increase antioxidant

Activity. Prevent

proliferation of cancer

- help speed excretion of

carcinogen from the body

21
Flavonoid Anthocyanine, Beans, citrus fruits - Block access of
Anthoxanthin
carcinogen, prevents

Malignant change in cells.

Prevent cancer

Saponins Panaxadiol, Potato, tomato, Reduce glucose and

glycerol
Panaxatriol soybean, beans
Uptake in the gut.

Phytosterols β-sitosterol, Potatoes, tomatoes, Block excess uptake of

dietary
campesterol, vegetable oils, alfafa
cholesterol and facilitate
stigmasterol sprout
cholesterol excretion

Terpenes Mono-terpenes Garlic, maize, ginger Help detoxify carcinogens,

inhibit

spread of cancer

Isothiocyanates Allylisothiocyana Cruciferous Suppress tumor growth,

te, boost
vegetables including
indoles,sulfurapha proliferation of cancer-
cabbage, curli
ne fighting
flower,
Enzymes.
broccoli

2.5.3 Phenolics / Polyphenols

Phenolic phytochemicals are the largest category of phytochemicals and the most widely
distributed in the plant kingdom. Structurally, they contain aromatic ring containing one or

22
more hydroxyl groups (Connell and Fox, 2001). Based on the number of carbon atoms
present in its structure, phenolics are categorized into five major groups.

i. C6 group: This group of phenol includes simple phenols and benzoquinones with
six carbon atoms.
ii. C6Cn group: Phenolic acid and hydroxycinnamic acid derivatives are included in
this group.
iii. C6CnC6 group: The largest group of phenolic compound includes flavonoids which
have low molecular weight and are further of five types (flavones, flavonols,
flavanols, flavanones and anthocyanins) on the basis of substitution pattern of
carbon ring.
iv. (C6Cn)3 group: This group consists of lignins and lignans.
v. Tannins: Tannins are high molecular weight phenols and classified into two main
categories (hydrolysable and condensed tannins) (Connell and Fox, 2001)

2.5.3.1 Significance of phenolic compounds

Phenolic compounds play various roles in plants, few of which can be listed below
(Connell and Fox, 2001):

 As antioxidant compounds: The main and most important role of phenol is their
antioxidant property. They act as free radical scavengers which are formed due to
high UV radiation.
 As structural polymers: Lignin is the most important and widely distributed
phenolic compounds which act as structural unit of plants.
 As defensive compounds: Due to presence of tannins, plant develops an astringent
taste. Tannins interact and precipitate with proteins which results in bitter taste of
plants. Consequently, they act as feed deterrents in most of the cases.
 As signal compounds: Many biochemical metabolic pathways have phenolic
compounds as their signal molecules. For instance, in salicylic acid pathway,
methyl salicylate (phenolic compound) acts as a signaling compound. Another
phenolic signaling compound reported is de-hydrodiconiferyl alcohol glucosidase
(DCG).

23
  As pollinator attractors: Simpe phenolic acids with low molecular weight are
responsible for aroma and attractive coloration of flowers which attract pollinating
agents. Phenols with these functions are anthocyanins, flavonoids etc.
 As UV screen: The phenolics present in plant cuticle play an important role in
screening the amount of UV radiations reaching earth through ozone layer.

2.5.4 Flavonoids

Flavonoids are the largest group of phenolic compounds and have a basic skeleton
composed of three rings (C6-C3-C6). They are classified into six major classes according
to their substitution pattern in the B- and C-rings, which are flavan-3-ols, anthocyanins,
flavones, isoflavones, flavanones and flavonols (Harbone and Baxter, 2000). The flavonoid
polymers are also known as proanthocyanidins. Flavonoids occur as plant secondary
metabolites that are involved in pigmentation, antioxidants, antimicrobials, antistressors,
and UV irradiation protection (Vaya and Aviram, 2001). More than 4000 flavonoids have
been described so far within the parts of plants normally consumed by humans and
approximately 650 flavones and 1030 flavanols are known (Ghasemzadeh et al., 2010).

Fig. 2.3 Structure of Flavonoid

Source: Hertog et al. (1992)

2.5.4.1 Biological activity of flavonoid

Flavonoids have gained recent attention because of their broad biological and
pharmacological activities. They have been reported to exert multiple biological property
including antimicrobial, cytotoxicity, anti-inflammatory as well as antitumor activities but
the best-described property of almost every group of flavonoids is their capacity to act as
powerful antioxidants which can protect the human body from free radicals and reactive

24
oxygen species. The capacity of flavonoids to act as antioxidants depends upon their
molecular structure. The position of hydroxyl groups and other features in the chemical
structure of flavonoids are important for their antioxidant and free radical scavenging
activities (Kelley et al., 2002).

The β ring hydroxyl configuration is the most significant determinant of scavenging of

ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Spevies) because it donates
hydrogen and an electron to hydroxyl, peroxyl, and peroxynitrite radicals, stabilizing them
and giving rise to a relatively stable flavonoids radical (Pandey and Kumar, 2013).

Mechanisms of antioxidant action may include

 Suppression of ROS formation either by inhibition of enzymes or by chelating trace

elements involved in free radical generation.
 Scavenging ROS and
 Regulation or protection of antioxidant defenses

Flavonoid action involves most of the mechanisms mentioned above. Some of the
effects mediated by them may be the combined result of radical scavenging activity and the
interaction with enzyme functions. Flavonoids inhibit the enzymes involved in ROS
generation, that is, microsomal mono-oxygenase, glutathione s-transferase, mitochondrial
succinoxidase, NADH oxidase, and so forth (Pandey and Kumar, 2013).

2.5.5 Tannins

Tannins are polyphenols sometimes called plant polyphenols although originally the name
tannin was given to the plant extracts exhibiting astringency, without knowing their
chemical structures (Haslam, 1989). The features distinguishing tannins from plant
polyphenols of other types are basically the properties of the former: binding to proteins,
basic compounds, pigments, large-molecular compounds and metallic ions, and also
antioxidant activities, etc (Okadu and Ito, 2013). These are widely distributed in plant
flora. They are phenolic compounds of high molecular weight. Tannins are soluble in
water and alcohol and are found in the root, bark, stem and outer layers of plant tissue.
They form complexes with proteins, carbohydrates, gelatin and alkaloids. On the basis of
their structural characteristics it is therefore possible to divide the tannins into four major

25
groups: Gallotannins, ellagitannins, complex tannins, and condensed tannins (Saxena et al.,
2014).

Gallotannins are all those tannins in which galloyl units or their meta-depsidic
derivatives are bound to diverse polyol-, catechin-, or triterpenoid units.

Ellagitannins are those tannins in which at least two galloyl units (C–C) are coupled to
each other, and do not contain a glycosidically linked catechin unit.

Condensed tannins are all oligomeric and polymeric proanthocyanidins formed by

linkage of C-4 of one catechin with C-8 or C-6 of the next monomeric catechin.

Complex tannins are tannins in which a catechin unit is bound glycosidically to a

gallotannin or an ellagitannin unit.

2.5.5.1 Activity of tannins

Tannins have diverse effect on biological system since they are potential metal ion
chelators, protein precipitating agents and biological antioxidants. Because of the varied
biological roles that tannins can play and because of the enormous structural variation, it
has become difficult to develop models that would allow an accurate prediction of their
effects in any system (Skowyra, 2014).

The tannin-containing plant extracts are used as astringents, against diarrhoea, as

diuretics, against stomach and duodenal tumors and as anti-inflammatory, antiseptic,
antioxidant and haemostatic pharmaceuticals (Dolara et al., 2005). In the food industry
tannins are used to clarify wine, beer, and fruit juices. Other industrial uses of tannins
include textile dyes, as antioxidants in the fruit juice, beer, and wine industries, and as
coagulants in rubber production (Gyamfi and Aniya, 2002). Recently the tannins have
attracted scientific interest, especially due to the increased incidence of deadly illnesses
such as AIDS and various cancers. The search for new compounds for the development of
novel pharmaceuticals has become increasingly important, especially as the biological
action of tannin-containing plant extracts has been well documented (Palavy and Priscilla,
2006)

26
2.5.6 Phytochemical metabolism in human

Most phytochemicals found in foods exist in a variety of forms which influence their
digestion and absorption. Most common ones are the polyphenols which exist as glycoside
conjugates. Some glycosides must be digested to aglycones (unconjugated forms) before
being absorbed. Some other forms of phytochemicals are thought to be absorbed in the
intestines without intensive digestion. The absorption of most phytochemicals is thought to
involve a carrier. Also, many glycosides are neither digested nor absorbed in the small
intestines. Such phytochemicals not absorbed in the small intestine have been shown to
undergo microbial degradation by colonic microflora (Ross and Kasum, 2002). The
bacteria hydrolyse the glycosides, generating aglycones which may undergo further
metabolism to form various aromatic compounds (Bradlow et al., 1999).

Once absorbed, most phytochemical metabolites get conjugated in the small intestine or
in the Liver (Rhodes, 1996). Conjugation most often involves methylation, sulfating or
glucunnidation. These conjugated metabolites are then bound to plasma proteins such as
albumin and are transported through the blood to various parts of the body The amount of
these conjugated metabolites in the plasma varies considerably with the type of polyphenol
consumed, the food source, and the amount ingested however, after consumption of
specific polyphenols, little is known about the metabolism of the different polyphenols in
the body, and also about what metabolites are present in the plasma (Briskin, 2000).
2.6 Antioxidants

Any substance which is capable of delaying, retarding or preventing the development of

the rancidity or other flavors deterioration due to oxidation is called antioxidant (Coppen,
1983). Oxidation reactions are chemical reactions that involve the transfer of electrons
from one substance to an oxidizing agent. Antioxidants can slow these reactions either by
reacting with intermediates and halting the oxidation reaction directly, or by reacting with
the oxidizing agent and preventing the oxidation reaction from occurring (Pokorny, 2007).
Much is known about antioxidants because of their functional importance, interest in
antioxidants is high to protect edible oils, their derived products and also when used in
food products to provide baking and culinary characteristics and nutritional benefits.
Antioxidants are substances that generally prevent, delay or retard the onset of rancidity in
food products due to oxidation of the unsaturated fatty acids incorporated in food products.

27
The use of antioxidants helps to extend the shelf life of a food, minimizes waste and
nutritional losses, and extends the scope of use of various fats/oils (Bhattacharya, 2003).

2.6.1 Necessity of use of antioxidants

Although techniques like vacuum packaging or packing under inert gas (to exclude
oxygen) and refrigeration/freezing reduce the rate of autoxidation, they are not always
applicable. Furthermore, it is a fact that little oxygen is needed to initiate and maintain
oxidative process and it is quite impossible or expensive to remove the least traces of air
from a food product. Antioxidants are generally effective, easily applied and inexpensive.
Other justification for need of an antioxidant use are- an antioxidant can extend the shelf
life of a food, reduce wastage and nutritional losses (oil soluble vitamins) and more
important it can widen the range of fats which can be used (Hamilton, 1989).

2.6.2 Mechanism of action

The antioxidants are active in lengthening the induction period in the process of oxidation
of fats, probably due to the absorption of the activating energy of the chain reaction that
result in the oxidation of antioxidants. An antioxidant act by reacting with free radicals
fatty acid (free radiator peroxy free radical) as they are formed, converting them back to
the original substrate and then by terminating the chain propagation (or initiation). Free
radicals of the antioxidant molecule are formed in the process, but the structure of an
antioxidant so such that these are relatively stable and do not have enough energy to react
with the fat to form further free radicals (Coppen, 1983). The process scheme (Dugan,
1986) is in Fig. 2.4.

An antioxidant (AH) apparently reacts with free radicals in following manner:

R• + AH --------------------RH + A•

RO• +AH --------------------- ROH +A•

ROO• +A• --------------------ROOH +A•

R• + A• ----------------------RA

RO• +A• --------------------------ROA

A• + ROO• -------------------- ROOA (Non radical product)

28
A• + A• -------------------------- AA (Non radical product)

Fig. 2.4 Mechanism of action of antioxidant

At high concentrations the antioxidant may have a peroxidant effect (Pokorny, 2007)
and one of the reactions may be as follows:

A• + RH ---------------------- AH + R•

The mode of action of all antioxidants (artificial or natural) is similar to the above
mechanism (Lee, 1971).The antioxidant should be added to the fat as early as if possible in
its life to produce the maximum effect. It follows that an antioxidant will only really be
effective if it is added during the initiation period (Lee, 1971).

2.6.3 Types of antioxidant

Antioxidants are available in both natural and synthetic forms which are discussed
separately below.

2.6.3.1 Synthetic (artificial) antioxidant

Most of the synthetic antioxidants used are phenolic compounds among which Butylated
Hydroxytoluene (BHT) and Butylated Hydroxyanisole (BHA), Tert Butylated
Hydroquinone (TBHQ), Propyl Gallate (PG) are common in use. A quantitative tolerance
limits for this synthetic antioxidant in Federal Regulations are limited not exceeds total
content of 0.02% of fat or oils either alone or in combination (Bauernfeind and Cort,
1974). TBHQ,PG, BHA and BHT are the most commonly used antioxidants in food
industries. In addition to being oxidizable, BHA and BHT are fat-soluble. Both molecules
are incompatible with ferric salts. In addition to preserving foods, BHA and BHT are also
used to preserve fats and oils in cosmetics and pharmaceuticals (Imida et al., 1983).

In recent years the possible toxicity of synthetic antioxidants has been investigated by
several workers. Butylated Hydroxyanisole (BHA) has been banned. Most antioxidants are
phenolic in structure and by the donation of a hydrogen atom to the acyl group of peroxy
radical, they form relatively stable radicals and non-radical products. Japan following
research by showed that the antioxidant promoted carcinogenesis in rats. Butylated
Hydroxytoluene has also been implicated as a promoter of tumors (Imida et al., 1983).

29
2.6.3.1.1 Tert-Butylhydroquinone

Tert-Butylhydroquinone (TBHQ) is a synthetic food grade antioxidant that is used to

stabilize foods, fats and vegetable oils against oxidative deterioration and thus extending
their storage life. The structure of TBHQ is given in Fig. 2.5.

TBHQ is certified as safe for human consumption. In many major developing

organizations like FDA (Food and Drug Administration), FSIS (Food Safety and
Inspection Service) and others permit the use of TBHQ or combinations with BHA or BHT
concentrations up to 0.02% by weight of the fat or oil content of the food (Burr and Burr,
1929).

Fig. 2.5 Chemical structure of TBHQ

Source: Burr and Burr (1929)

2.6.3.1.2 Butylated Hydroxyanisole (BHA)

Butylated hydroxyanisole (BHA) is a mixture of two isomeric organic compounds, 2-

tertbutyl-4– hydroxyanisole and 3 – tert – butyl – 4 – hydroxyanisole. It is prepared from
4- methoxyphenol and isobutylene. It is a white or pale yellow solid (Crystal or Flake) with
a faint aromatic odor (Burr and Burr, 1929).

Fig. 2.6 Chemical structure of BHA

Source: Burr and Burr (1929)

30
2.6.3.1.3 Butylated Hydroxy Toluene (BHT)

Butlyated Hydroxyanisole is a synthetic antioxidant that is a commonly used fat soluble

food preservative since 1947, with broad biological activities. It prevents spoilage by
reacting with oxygen. It slows development of off-flavors, odors and color changes caused
by oxidation. It protects animals against radiation and the acute toxicity of various
xenobiotics and mutagens. Butylated hydroxyanisole (BHA) is a mixture of two isomeric
organic compounds, 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. It is
prepared from 4-methoxyphenol and isobutylene. It is normally insoluble in water, but for
commercial applications, it can be converted to a soluble form. BHT was first used as an
antioxidant food additive in 1954. An antioxidant is a substance that prevents the oxidation
of materials with which it occurs. BHT, therefore, prevents the spoilage of food to which it
is added. BHT has grown to be very popular. Among food processors and is now used in a
great range of products that include breakfast cereals, chewing gum, dried potato flakes,
enriched rice, potato chips, candy, sausages, freeze-dried meats, and other foods containing
fats and oils. BHT is sometimes used in conjunction with a related compound, Butylated
hydroxyanisole (BHA) as a food additive. BHT does have other commercial uses, as in
animal feeds and in the manufacture of synthetic rubber and plastics, where it also acts as
an antioxidant (Bishov and Henic, 1977).

Fig. 2.7 Chemical structure of Butylated Hydroxy Toluene

Source: Burr and Burr (1929)

2.6.3.2 Natural Antioxidant

Antioxidants are widely distributed in nature. Natural antioxidants impart no adverse effect
in its long run of use; and do not have a quantitative tolerance limit in Federal

31
Regulations.It imparts no adverse effect in its long run of use; natural antioxidants do not
have a quantitative tolerance limit in federal regulations (Bauernfeind and Cort, 1974).
Modern lipid science discovered that antioxidants are widely distributed in nature (Burr
and Burr, 1929). Natural antioxidants seems to be more adequate for protection against
oxidation and have many inherent qualities unsuppressed by the synthetic antioxidants
(Loliger, 1983).

Natural antioxidants can be used in a number of applications even where there is no

choice for anything else, because of company policy or food legislations and public
pressure group. There are some scientific evidences which alone is sufficient to support for
using natural antioxidants. The antioxidants activity from natural sources has been
demonstrated in spices (Chang et al., 1977); vegetable extracts (Pratt and Watts, 1964) and
plant protein and their hydrolysates (Bishov and Henic, 1977). None have achieved
commercial importance. The tocopherols are an important group of natural antioxidants.
(Chipault et al., 1952) and Aoes Jorgensen (1962) cover some its early work on tocopherol
in vitro animal fat oxidation study showed that tocopherol have antioxidant activity at
levels as low as (0.008%or 0.01%). Mixed with citric or ascorbyl palmate, tocopherol are
efficient as BHT. In absence of oxygen, they are relatively heat and light stable
(Bauernfeind and Cort, 1974).

The tocopherols are slightly viscous pale yellow liquids freely soluble in most
organicsolvents; insoluble in water. To retard the development of oxidative rancidity and
tocopherolsare used in foods as antioxidants. Tocopherols have a molecular wt. of 30-69
and boiling point 200-220oC (0.1 mm). In addition to food uses vitamin is used in food
supplement and pharmaceutical dosage formulation.Tocopherols are readily oxidized and
consequent protects the fat from oxidation (Meyer, 1987). Mode of action of natural
antioxidants are not different from dose of similar synthetic phenols and polyphenols are
proton donors which terminate free radical chains (Bishov and Henic, 1977).

2.6.3.3 Synergistic Antioxidants

The preventive antioxidants which act by reducing the rate of chain initiation is called
synergistic antioxidants although they have no effect as protectants when used along with
fats (Lee, 1971).These compounds helps to increase (improve) the ability of the phenolic
antioxidants to retard rancidity. Furia (1968) has presented an excellent review of the use

32
of sequestrates (metal in activators) in foods. Many components exhibit metal deactivating
properties in edible triglyceride oils as evidenced by improvement in oxidative and/or
flavor stability. Among these most important is citric acid (Dutton et al., 1949).

All metal inactivating compounds have free hydroxyl for carboxyl groups that co-
ordinate readily with metal forms salts readily proposed the metal inactivators, in effect
complexes with peroxidant metal and hold them in a chelat or ring structure held winter
co-ordination complexes, where the metal can no longer function as peroxidant (Cowan,
1999).

2.6.4 Oxidative stress

The term is used to describe the condition of oxidative damage resulting when the critical
balance between free radical generation and antioxidant defenses is unfavorable (Rock et
al., 1996). Oxidative stress, arising as a result of an imbalance between free radical
production and antioxidant defenses, is associated with damage to a wide range of
molecular species including lipids, proteins, and nucleic acids (McCord, 2000). Short-term
oxidative stress may occur in tissues injured by trauma, infection, heat injury, hypertoxia,
toxins, and excessive exercise. These injured tissues produce increased radical generating
enzymes (e.g., xanthine oxidase, lipogenase, cyclooxygenase) activation of phagocytes,
release of free iron, copper ions, or a disruption of the electron transport chains of
oxidative phosphorylation, producing excess ROS. The initiation, promotion, and
progression of cancer, as well as the side-effects of radiation and chemotherapy, have been
linked to the imbalance between ROS and the antioxidant defense system. ROS have been
implicated in the induction and complications of diabetes mellitus, age-related eye disease,
and neurodegenerative diseases such as Parkinson's disease (Rao et al., 2006).

2.6.5 Mechanism of action of antioxidants

Two principle mechanisms of action have been proposed for antioxidants (Rice-Evans and
Diplock, 1993). The first is a chain- breaking mechanism by which the primary antioxidant
donates an electron to the free radical present in the systems. The second mechanism
involves removal of ROS reactive nitrogen species initiators (secondary antioxidants) by
quenching chain-initiating catalyst. Antioxidants may exert their effect on biological
systems by different mechanisms including electron donation, metal ion chelation, co-
antioxidants, or by gene expression regulation (Krinsky, 1992).
33
2.6.6 Levels of antioxidant action

The antioxidants acting in the defense systems act at different levels such as preventive,
radical scavenging, repair and de novo, and the fourth line of defense, i.e., the adaptation.
The first line of defense is the preventive antioxidants, which suppress the formation of
free radicals. Although the precise mechanism and site of radical formation in vivo are not
well elucidated yet, the metal-induced decompositions of hydroperoxides and hydrogen
peroxide must be one of the important sources. To suppress such reactions, some
antioxidants reduce hydroperoxides and hydrogen peroxide beforehand to alcohols and
water, respectively, without generation of free radicals and some proteins sequester metal
ions (Lobo et al., 2010).

Glutathione peroxidase, glutathione-s-transferase, phospholipid hydroperoxide

glutathione peroxidase (PHGPX), and peroxidase are known to decompose lipid
hydroperoxides to corresponding alcohols. PHGPX is unique in that it can reduce
hydroperoxides of phospholipids integrated into biomembranes. Glutathione peroxidase
and catalase reduce hydrogen peroxide to water (Lobo et al., 2010).

The second line of defense is the antioxidants that scavenge the active radicals to
suppress chain initiation and/or break the chain propagation reactions. Various endogenous
radical-scavenging antioxidants are known: some are hydrophilic and others are lipophilic.
Vitamin C, uric acid, bilirubin, albumin, and thiols are hydrophilic, radical-scavenging
antioxidants, while vitamin E and ubiquinol are lipophilic radical-scavenging antioxidants.
Vitamin E is accepted as the most potent radical-scavenging lipophilic antioxidant (Lobo et
al., 2010).

The third line of defense is the repair and de novo antioxidants. The proteolytic
enzymes, proteinases, proteases, and peptidases, present in the cytosol and in the
mitochondria of mammalian cells, recognize, degrade, and remove oxidative modified
proteins and prevent the accumulation of oxidized proteins (Lobo et al., 2010).

The DNA repair systems also play an important role in the total defense system against
oxidative damage. Various kinds of enzymes such as glycosylases and nucleases, which
repair the damaged DNA, are known. There is another important function called adaptation
where the signal for the production and reactions of free radicals induces formation and
transport of the appropriate antioxidant to the right site (Lobo et al., 2010).
34
 Part III

Materials and methods

3.1 Raw materials

The plant under study during the research was Moringa oleifera leaves. It was collected
from the Tamor River banks of Dhankuta District.

3.2 Equipment and chemicals

The following equipment and chemicals used were available in campus. The list of
chemicals used for the analysis is shown in Table 3.1 and the list of equipment is shown in
Table 3.2.

Table 3.1 List of chemicals used:

Chemicals Supplier/Manufacturer Other specifications

Sodium hydroxide (NaOH) Thermo fisher Scientific India Pellets, AR grade, 98%
Pvt. Ltd.

Hydrochloric acid (HCl) Thermo Electron LLS India Pvt. 36%, LR grade
Ltd.

Sulphuric acid (H2SO4) Thermo fisher Scientific India 97%, LR grade

Pvt. Ltd.

Boric acid Merck (India) Limited Amorphous

Oxalic acid Merck (India) Limited Crystal

Petroleum ether Merck life Pvt. Ltd. B.P. 60℃-80℃

Sodium Carbonate Qualigens fine chemicals 99.5%, LR grade

(Na2CO3)

Sodium bicarbonate - -
(NaHCO3)

36
Methanol Merck life science Pvt. Ltd 99% Liquid

Ethanol Mt. Everest Industrial Works

Guaicol High media 99% Liquid

H2O2 solution Thermo electron LLS India Pvt. 30%

Ltd

Sodium nitrate(NaNO2) Thermo Fischer scientific India, 98%

Pvt. Ltd

Aluminum S.D fine-chem Ltd 98% Hygroscopic

Chloride(AlCl3)

Ferric chloride(FeCl3) Thermo Fischer scientific India, 96% Anhydrous

Pvt. Ltd

Chloroform Merck life science Pvt. Ltd 99% Liquid

Folin-ciocalteu„s reagent Thermo Fischer scientific India, Liquid

Pvt. Ltd

Acetic acid Thermo Fischer scientific India, 99% Liquid

Pvt. Ltd

Gallic acid - -

Ninhydrin solution Central drug house Pvt. Ltd Powder

DPPH Himedia laboratories(India) Pvt. Amorphous

Ltd

37
Table 3.2 List of equipment used

S.N Physical apparatus Specification

1. Electric balance Phoenix instrument, 620g

2. Spectrophotometer Labtronics, India

3. Soxhlet apparatus Y.P. Scientific industries

4. Hot air oven Victolab, India

5. Incubator Victolab, India

6. Muffle furnace Accumax, India

7. Knives: Stainless steel knives were used for the purpose of cutting

8. Micropipette, pipette

9. Desiccator

10. Thermometer

11. Measuring cylinder

12. Refrigerator

13. Mortar and pestle, Grinder, bamboo pinchers etc.

14. Steam cooker

15. Glassware (Beaker, Volumetric flask, conical flask, Burette, Petridish, Porcelain basin,
Crucible etc.)

16. Kjeldahl digestion and distillation set

3.4 Collection and preparation of sample

A common variety of Moringa oleifera collected from Dhankuta district Tamor River,
Nepal. The leaf samples were divided into four portions:

 Fresh Leaves: Washed and drained leaves. Stored at 4o C until analyzed.

38
  Water blanched leaves: Washed and drained leaves, water blanched at 98o C for 2
min in a cooker. After that blanched leaves were dried in a cabinet drier at about
50o C.
 Cabinet dried leaves: Fresh leaves were dried in a cabinet drier at about 50o C for
about 5 h.
 Dry roasted: Fresh leaves were dried in a frying pan at 120oC for 4 min.

Samples were dried to final moisture content below 6%.

From the collected fresh sample 10 g of plant leaf was crush into powder and then
powdered plant materials were steeped in 80% methanol (100 ml) for 12 h at room
temperature. They were then filtered through Whatman No. 41 filter paper. Finally,
extracts were transferred to brown colored glass bottles, sealed by using caps and stored at
4±2oC until analysis. Similarly for dried sample, blanched dried sample and pan dried
sample methanolic extract were also prepared and stored. The crude extract so obtained
were analysed for total phenol contents, flavonoids, tannins, chlorophylls and DPPH free
radical scavenging activities. The flow diagram of methodology is shown in Fig. 3.1.

39
 Fresh leaves

Cleaning

Sorting/Grading

Blanching Cabinet drying Dry roasting

Moringa powder

Proximate Analysis Phytochemical analysis Sensory analysis

Protein Total phenol content Color
Fat Total flavonoid content Flavor
Crude fiber Tannin Taste
Ash content After taste
Antioxidant activity
Moisture Overall acceptability
Chlorophyll
Vitamin C

Fig. 3.1 Flow diagram of methodology

3.5 Preparation of plant extracts

Plant materials were extracted as per with slight modification. Briefly, 10 g of powdered
plant materials were stepped in 80% methanol (100 ml) for 12 h at room temperature. They
were then filtered through Whatman no. 41 filter paper. Finally, extracts were transferred
to brown colored glass bottles, sealed by using caps and stored at 4 ± 2oC until analysis.
The extract concentration was determining by evaporating 5 ml of extract (at 80oC) to
dryness and measuring the weight (Ahmad et al., 2013).

3.6 Phytochemical qualitative analysis

The plant methanolic extracts were screened for the presence of the phytochemical classes
by using the standard following methods (Jaradat et al., 2015).

a. Tests for proteins

Ninhydrin test: Boil 2 ml of 0.2% Ninhydrin solution with the entire plant Crude extract,
appeared violet color indicate the presence of proteins and amino acids.
40
b. Tests for carbohydrates

 Fehling„s solutions test: Boil a mixture of Fehling solutions A and B with equal
volumes were added to crude plant extract. A red color precipitate indicated the
presence of reducing sugars.
 Benedict„s reagent test: Boil 2 ml of Benedict„s reagent with a crude extract, a
reddish brown color indicated the presence of the carbohydrates.
 Molisch„s solution test: Shake 2 ml of Molisch„s solution with crude plant extract
then add 2 ml of H2SO4 concentrated and poured carefully along the side of the test
tube a violet ring appeared at the inter phase of the test tube indicated the presence
of carbohydrate.
 Iodine test: 2 ml of iodine solution mixed with crude plant extract. Purple or dark
blue colors prove the presence of the carbohydrate.

c. Test for phenols and tannins:

 Two milliliter of 2% solution of FeCl3 mixed with crude extract. Black or blue-
green color indicated the presence of tannins and phenols.

d. Tests for flavonoids

 Shinoda test: pieces of magnesium ribbon and HCl concentrated were mixed with
crude plant extract after few minutes pink colored scarlet appeared that indicated
the presence of flavonoids.
 Alkaline reagent test: 2 ml of 2% NaOH solution was mixed with plant crude
extract, intensive yellow color was formed, which turned into colorless when added
2 drops of diluted acid to solution, this result indicated the presence of flavonoids.

e. Test for saponins

Five milliliter of distilled water was added to crude plant extract in a test tube and it was
shaken vigorously. The foam formation indicated the presence of saponins.

41
f. Tests for glycosides

 Liebermann„s test: 2 ml of acetic acid and 2 ml of chloroform mixed with entire

plant crude extract. The mixture was then cooled and added H2SO4 concentrated,
green color indicated the entity of aglycone steroidal part of glycosides.
 Salkowski„s test: H2SO4 concentrated (about 2 ml) was added to the entire plant
crude extract. A reddish brown color produced indicated the entity of steroidal
aglycone part of the glycoside.
 Keller-kilani test: A mixture of acetic acid glacial (2 ml) with 2 drops of 2% FeCl3
solution was added to the plant extract and H2SO4 concentrated. A brown ring
produced between the layers which indicated the entity of cardiac steroidal
glycosides.

g. Test for steroid

Two milliliter of chloroform and concentrated H2SO4 were mixed with the entire plant
crude extract. In the lower chloroform layer produced red color that indicated the presence
of steroids. Another test was performed by mixing 2 ml of each of acetic acid with H2SO4
concentrated and crude extract with 2 ml of chloroform. Green color indicated the entity of
steroids.

h. Test for terpenoids

Two milliliter of chloroform was mixed with the plant extract and evaporated on the water
path then boiled with 2 ml of H2SO4 concentrated. A grey color produced indicated the
entity of terpenoids.

3.7 Quantitative analysis

3.7.1 Determination of moisture content

Moisture content of the sample was determined by hot air oven method as standard method
of (Ranganna, 2007).

42
3.7.2 Determination of crude protein

The crude protein was determined by using Kjeldahl„s method. 2 g fat less samples was
digested; steam distillated after decomposing the former NaOH. Titration of entrapped
NH3 boric acid was done with standard acid as standard method of (Ranganna, 2007).

3.7.3 Determination of crude fat

The fat content was determined by Soxhlet method. Solvent extraction of 10 g sample was
done by recycling hot solvent for number of times until complete extraction and fat was
recovered by evaporating away the solvent as standard method of (Ranganna, 2007).

3.7.4 Determination of ash content

Ash content was determined using muffle furnaces according to Ranganna (2007). 5 g of
weighed sample in silica crucible was charred in hot plate till no smoke raise from it and
finally, ashing was done in muffle furnace at 550oC to the constant weight. The difference
in weight was the total ash content remaining in crucible, under standardized condition.

3.7.5 Determination of crude fiber

Crude fiber was determined by using chemical process, the sample was treated with boiling
dilute Sulphuric acid, boiling sodium hydroxide and then with alcohol as standard method
of Ranganna (2007).

3.7.6 Determination of total phenol

Total phenolic content (TPC) in the plant methanolic and ethanolic extracts was
determined using spectrophotometric method (Jaradat et al., 2015) with some
modifications. The reaction mixture was prepared by mixing 0.5 ml of plant extract
solution, 2.5 ml of 10% Folin Ciocalteu„s reagent dissolved in water and 2.5 ml of 7.5% of
Na2CO3 aqueous solution. The samples were thereafter incubated in a thermostat at 45ºC
for 45 min. The absorbance was determined using spectrophotometer at wave length 765
nm. The samples were prepared in triplicate for each analysis and the mean value of
absorbance was obtained. The same procedure was repeated for the standard solution of
gallic acid and the calibration line was construed. Based on the measured absorbance, the
concentration of gallic acid equivalent expressed in terms of mg of GA/g of extract.

43
3.7.7 Determination of flavonoids

Total flavonoid content was determined using a modified aluminum chloride assay method
as described by (Barek and Hasmadi, 2015). 2 ml of solution was pipette out in a test tube
in which 0.2 ml of 5% Sodium Nitrate (NaNO3) was mixed and stand for 5 min. 0.2 ml
Aluminum Chloride (AlCl3) was pipetted out, mixed in the tube and allowed to stand for 5
min. This followed addition of 2 ml of 1 N Sodium Hydroxide (NaOH) in the tube and
finally volume was made up to 5 ml. The absorbance was measured after 15 min at 510 nm
against a reagent blank. The test result was correlated with standard curve of Quercetin
(20, 40, 60, 80, 100 μg/ml) and the total flavonoid content is expressed as mg quercetin
equivalents (QE) (Barek and Hasmadi, 2015).

3.7.8 Determination of tannins

Tannin was determined by Folin-ciocalteu method. About 0.1 ml of the sample extracts
added to volumetric flask(10ml) containing 7.5 ml distilled water and 0.5 ml folin-
ciocalteu reagent,1 ml 35% Na2Co3 solution and dilute to 10 ml distilled water. The
mixture is shaken well and kept at room temperature for 30 min. A set of reference
standard solution of Gallic acid (20, 40, 60, 80, 100 μg/ml) are prepared in same manner as
described earlier. Absorbance for test and standard solution are measured against blank at
725 nm with an UV/visible spectrophotometer. The tannin content is expressed in terms of
mg of GAE/g of extract (Mythili et al., 2014).

3.7.9 Determination of chlorophyll

Total chlorophyll content in moringa sample is determined as per (Rai, 2006)

Chl a, mg/g tissue = 12.7(A663) -2.69(A645) × V/1000×W

Chl b, mg/g tissue = 22.9(A663) -4.68(A645) × V/1000×W

Total chlorophyll, mg/g tissue = Chl a + Chl b (calculated above)

Where A = absorbance at specific wavelengths

V = final volume of chlorophyll extract

W = fresh weight of tissue extracted

44
3.7.10 Determination of Ascorbic acid (vitamin C)

Vitamin C in leaves sample were determined by usual titration method as per Ranganna
(2007).

The vitamin C content in the sample was calculates as follows:

( )
( ) ( )

3.7.11 Determination of blanching time

100 g of moringa leaves were tied up loosely and dipped in boiling water. The blanching
time was ranged from 30 s of interval from 1-5 min. Each sample was grounded in clean
mortar and pestle with equal volume of water. The ground content was strained through
filter paper in another clean test tube. About 5 ml of content was taken and to it 1 ml of 1%
guaicol solution (in 95% alcohol) and 1 ml 0.5% of H2O2 added. The tube was allowed to
stand for 5 min. The development of brown color in the content indicates the presence of
peroxidase, the most heat resistant enzyme.

3.7.12 Determination of DPPH radical scavenging activity

DPPH free radical scavenging activities (antioxidant activities) of extracts were determined
by method described by (Vignoli et al., 2011) with slight variation. Different dilutions of
the extracts were made using 80% methanol. Then 1 ml of the extract was mixed with 2 ml
of 0.1 mM DPPH solution. The absorbance was read at 517 nm after 30 min incubation in
the dark. Finally, percentage scavenging activity was determined using following equation

% scavenging activity = (Ac-As) × 100 /Ac

Where Ac is the absorbance of control and As is the absorbance of test sample.

3.8 Sensory analysis

Sensory analysis of the moringa leaf powder was analyzed as similar to tea tasting
procedure developed by TRA (Tea Research Association), Assam. 2.8 g of Moringa
oleifera samples were weighed and they were kept in a cup. 140 ml water was added and
covered with lid. It was left for 5 min. The brew was then transferred to the bowl and the

45
infusion was taken out in the lid. Brew was evaluated for sensory attributes (color, flavor,
taste, after taste and overall acceptability).

3.8 Optimization of moringa Powder

After the preparation of extract with 80% methanol, phytochemical analysis for four
sample of Moringa oleifera was done, finally sensory analysis for the prepared sample of
Moringa oleifera was carried out as per 9-point hedonic rating to optimize color, flavor,
taste¸ after taste, and overall acceptability. Criteria of optimization for Moringa oleifera
leaves are shown in Table 3.3 and Table 3.4.

Table 3.3 Optimization of phytochemical parameters

Parameters Goals

TPC Maximize

TFC Maximize

Tannin Minimize

DPPH Scavenging activity Maximize

Chlorophyll Maximize

Table 3.4 Optimization of sensory parameters

Parameters Goals

Color Maximize

Flavor Maximize

Taste Maximize

After Taste Maximize

Overall acceptability Maximize

46
3.9 Statistical analysis

Analyses were carried out in triplicate. Statistical calculations were performed in Microsoft
office Excel 2010. All the data obtained in the experiment were analyzed for significance
by Analysis of Variance (ANOVA) using statistical software Genstat Release v12 (Payne
et al., 2009). From this, means were compared using Fischer‟s protected LSD (Least
Significance Difference) at 5% level of significance.

47
 Part IV

Results and discussion

For the preparation Moringa Oleifera leaves were collected from Dhankuta district, Tamor
River banks, Nepal. The leaves were packed in black polyethene bags to prevent the
oxidation of vitamin C and the chlorophyll of the leaves. Then, leaves were washed with
clean water to remove the dirt adhered in it. The stems of leaves were removed and fresh
leaves were subjected to blanching, drying and dry roasting and their phytochemical
composition, chlorophyll and vitamin C were analyzed.

4.1 Physicochemical properties of moringa Leaves

The physiochemical properties of fresh moringa leaves are shown in Table 4.1. The
moisture content, crude protein, crude fiber, fat content, ash and vitamin C for fresh
Moringa oleifera leaves was found to be 75.6%, 24.85%, 8.19%, 7.10%, 8.72% and 155.67
mg/100g respectively. The results for physiochemical properties of Moringa oleifera
leaves are in correlation with (Kshirsagar et al., 2017; Nambiar et al., 2003).

The physiochemical properties of blanched, dried and roasted moringa powder are
presented in Table 4.1. The moisture content, crude protein, fat, crude fiber, ash content
and vitamin C content of blanched moringa leaves was found to be 6.28%, 22.86%, 8.43%,
7.7%, 7.34% and 78.10 mg/100 g. Similarly for cabinet dried leaves were found to be
5.95%, 27.67%, 8.23%, 9.4%, 7.55% and 55.66 mg/100 g and finally for dried roasted
were 6.54%, 20.36%, 8.9%, 9.48%, 7.65% and 49.66 mg/100 g respectively.

48
Table 4.1 Physicochemical properties of fresh, blanched dried, cabinet dried and roasted
moringa leaves

Parameter Fresh Leaf Blanched dried Cabinet dried Dry Roasted

Moisture % 75.6±0.86ab 6.28±0.04ab 5.95±0.50a 6.54±0.14b

Protein % 24.85±0.57b 22.86±0.11b 27.67±0.28c 20.36±0.11a

Crude fiber % 8.19±0.04c 7.7±0.17a 9.40±0.17b 9.48±0.01b

Fat % 7.10±0.22a 8.43±0.05a 8.23±0.05a 8.90±0.1b

Ash % 8.72±0.11b 7.34±0.14a 7.55±0.09b 7.65±0.05b

Vitamin C (mg/100 g) 155.67±1.15c 78.10±0.85c 55.66±0.57b 49.66±0.57a

F pr. ≤.001 ≤.001 ≤ .001 ≤ .001

*data represent mean ± S. D. of triplicate analysis

Drying reduces the moisture content of Moringa oleifera leaves from 75% to less than
10%. The leaves can be thus preserved without microbial contamination. Neither blanching
nor drying methods had any significant effect on the macronutrient composition of
moringa oliefera leaves. The protein content of Moringa oleifera leaves and leaf powders
ranged between 20.36% to 27.55% on dry basis. The fat content of moringa oliefera leaves
is between 7.1% to 8.8% and they have relatively high fiber content up to 10% in dried
leaves. The ash content varied from 7.6% to 8.7% indicating the moringa leaves are rich in
minerals compounds. Moringa oleifera leaves contain high levels of proteins (20-28%) and
relatively low level of lipids content (7-8%). According to (Amaglo, 2010) and (Sanchez-
machado et al., 2010), these lipids are essentially made up of unsaturated fatty acids such
as linolenic acid (51-57%). The slight decreases in protein content of leaves is probably
due to leaching where loss of water soluble nitrogen containing compounds such as free
amino acids and nucleotides might have occurred during the process (Njoroge et al., 2015).
Blanching treatment decreases the fiber content of the leaves whereas blanching had very
limited effect on fat content of moringa leaves which is similar with finding of (Nkafamiya
et al., 2010). This observation in addition to its antioxidant activity reinforces the
potentials of Moringa oleifera leaves as ingredient in the formulation of functional foods.

49
The decrease in ash content with blanching could be due to the fact that the heat weakens
cell membrane in moringa tissue and thus some minerals could have been leached out by
boiling water (Ferracane et al., 2008).

The Vitamin C content of fresh Moringa oleifera was found to be 155 mg/100 g and
this was significantly reduced by blanching to 78 mg/100g. Cabinet dried and dry roasting
had a most destructive effect on vitamin C content, reducing it to 55 mg/100 g and 50.67
mg/100 g respectively. Blanching prior to drying had a protective effect on the destruction
of vitamin C, as blanched dried samples had higher residual vitamin C contents than their
unblanched counterparts. Dry roasting decreases the most vitamin C compared to
blanching and cabinet drying.

The decrease in vitamin C with blanching and drying is in accordance with the fact that
it is heat liable vitamin easily oxidized on exposure to air and heat (Gupta et al., 2013;
Oyetade et al., 2012) and is also water soluble. In accordance with our observation (Joshi
and Mehta, 2010) have equally observed greater vitamin C losses in cabinet drying of fresh
leaves compared to blanching while losses of vitamin C with blanching have been reported
(Adefegha and Oboh, 2011; Mutiara et al., 2012).

4.2 Optimization of blanching time

Fruits and vegetables are heat treated or blanched to minimize deteriorative reactions or to
inactivate the enzymes. Blanching of fruits and vegetables are done either in hot water,
steam or selected chemical solutions (Luna-Guzman and Barret, 2000; Severini et al.,
2004).

Proper combination of time and temperature during processing methods such as blanching
or microwaving is important in order to minimize quality loss during processing. These
methods might cause undesirable changes on the physicochemical properties, such as
color, texture or bioactive compounds, on account of heat-induced diffusion or leaching
losses. Thus, it is important to optimize the time and temperature of any processing method
in order to achieve minimal loss of quality (Gupta et al., 2012). The blanching time-
temperature optimization result for moringa leaf

50
Table 4.2 Blanching time-temperature optimization for moringa leaves

Table 4.2.a Catalase test at different time-temperature

Blanching time (min) 0 1 1.5 2 2.5 3 3.5 4 4.5 5

Catalase test for sample at 90◦C + + + + + + + - - -

Catalase test for sample at 91◦C + + + + + + - - - +

Catalase test for sample at 92◦C + + + + + - - - - -

Catalase test for sample at 93◦C + + + + - - - - - -

Catalase test for sample at 94◦C + + + - - - - - - -

Catalase test for sample at 95◦C + + - - - - - - - -

Catalase test for sample at 96◦C + + - - - - - - - -

Catalase test for sample at 97◦C + - - - - - - - - -

Catalase test for sample at 98◦C + - - - - - - - - -

Table 4.2.b Peroxidase test at different time-temperature

Blanching time (min) 0 1 1.5 2 2.5 3 3.5 4 4.5 5

Peroxidase test for sample at 90◦C + + + + + + + + + +

Peroxidase test for sample at 91◦C + + + + + + + + + +

Peroxidase test for sample at 92◦C + + + + + + + + - -

Peroxidase test for sample at 93◦C + + + + + + + - - -

Peroxidase test for sample at 94◦C + + + + + + + - - -

Peroxidase test for sample at 95◦C + + + + + + - - - -

Peroxidase test for sample at 96◦C + + + + + - - - - -

Peroxidase test for sample at 97◦C + + + + - - - - -

51
Peroxidase test for sample at 98◦C + + + - - - - - - -

Therefore, from the table optimum blanching time-temperature for the moringa leaf was
found to be 2 min at boiling water i.e. 98oC. At this blanching condition, the peroxide
enzymes responsible for browning reactions gets deactivated which was observed by
peroxidase visual test method and high temperature in short time treatment is also
effective. Similar results were observed by (Kaushal et al., 2013; Odedeji et al., 2014) in
blanching taro leaves.

4.3 Qualitative phytochemical screening of Moringa oleifera leaves

During the experimental work, the methanol extract of Moringa oleifera leaves shown to
have the total phenol, total flavonoid, tannin and antioxidant activity which is shown in
Table 4.3

Table 4.3 Qualitative analysis for phytochemicals

Test Result

Proteins +ve

Carbohydrates +ve

Phenols and Tannins +ve

Flavonoid +ve

Saponins +ve

Glycosides +ve

Steroid -ve

Terpenoids -ve

Plus (+) =positive test; minus (-) =negative test

The phytochemicals screening of methanol extract of Moringa oleifera showed that the
leaves were rich in carbohydrates, phenol, tannins, flavonoids, saponins and glycosides but
not in proteins, steroid and terpenoids. Similar result for phytochemicals screening in

52
Moringa oleifera leaves was obtained (Isituo and Jaramilo, 2015; Otieno et al., 2016;
Unuigbe et al., 2014).

4.4 Quantitative analysis of phytochemicals in Moringa oleifera leaves

Treatment A, B, C and D refers to fresh, water blanched, cabinet dried and dry roasted
leaves respectively as shown in Table 4.4

Table 4.4 Quantitative analysis of phytochemicals in Moringa oleifera leaves

Treatments TPC TFC Tannins DPPH Chlorophyll

radical
(mgGAE/g) (mgQE/g) (mgGAE/g) (mg/g)
Scavenging
activity (%
inhibition)

A 146.0±5.5b 526.7±95.7c 12.81±0.62d 82.98±0.75d 23.27±1.44c

B 93.9±10.1a 453.5±28.5bc 8.36±0.46b 64.63±0.45c 17.32±0.02b

C 90.2±3.5a 377.4±31.8b 9.64±0.46c 60.00±0.87b 17.28±0.02b

D 89.2±0.4a 270.0±35.9a 6.39±0.36a 52.73±0.88a 7.15±0.10a

F pr. ≤ .001 ≤ .001 ≤ .001 ≤ .001 ≤ .001

*Values means are triplicate results, figures in the parenthesis are the standard deviations.
Figures with same superscript are not significantly different. Figures with different
superscript are significantly different.

4.4.1 Effect of processing conditions on total phenol content

4.4.1.1 Effect of blanching on TPC

The TPC content of blanched Moringa oleifera leaves were determined as per the standard
provided. During blanching the phenol content decreased from 146.0 mg/g to 93.9 mg/g.
The statistical analysis (One-way ANOVA) showed that the phenol and flavonoid content
was significantly decreased (p<0.05) during blanching as compared to fresh moringa
leaves.

53
 Irondi et al. (2016) reported that the decreases in phenolic compounds in blanched
leafy vegetables and the decrease was attributed due to leaching out of water soluble
phenolic compounds. 70% methanol extracted the maximum phenolic and flavonoid
content occurred in the moringa leaves compared to 70% ethanol and water as reported by
(El sohaimy et al., 2015). The decreasing nature of phenol content during blanching is also
reported by (Amin and Lee, 2005) and (Nobosse et al., 2017). Loss in phenolic content of
vegetables due to blanching is reported by (Gupta et al., 2012). And this may be due to the
oxidation of the phenolic or their leaching into water during blanching (Roy et al., 2009). It
is known that phenolic compounds are occurring in soluble forms and in combination with
cell wall components in plants. Thus disruption of the cell walls and the breakdown of the
phytochemicals occur due to high temperature of the blanching there by leading to their
leaching into blanching water (Francisco et al., 2010).

4.4.1.2 Effect of drying on TPC

The total phenol content dried Moringa oleifera leaves were determined as per the standard
provided. During drying of moringa leaves, the total phenol content decreased from 146
mg/g to 90.2 mg/g. The statistical analysis showed that there is significant difference in
TPC of fresh and dry leaves. The observation for the total phenol content in fresh leaves is
closer to the observation made by (Iqbal and Bharger, 2006).

The percentage loss in phenol content was found to be 77.27% which was closer to the
observation done by (Zhang et al., 2009). According to Mrad et al. (2012) the decreases in
TPC during drying could be attributed to the binding of polyphenols which cannot be
extracted or determined by available methods. Ancos et al. (2000) also reported that the
polyphenols compound may also deteriorate depending upon heat treatment.

4.4.1.3 Effect of dry roasting on TPC

The total phenol content of fresh Moringa oleifera was found to be 146 mg/g. During dry
roasting of leaves, the TPC decreased from 146 mg/g to 89.2 mg/g. The statistical analysis
(one-way ANOVA) showed that the TPC of moringa was significantly decreased (p<0.05)
during dry roasting. It was noted that, roasted moringa leaves showed lower concentration
on TPC than other processing methods.

54
 The losses in total phenol content during dry roasting might be due to the process
conditions in particular, the temperature and the duration used (Michalczyk et al., 2009;
Schieber et al., 2001). The integrity of all structure is affected by thermal breakdown by
various chemical reactions involving enzymes, light and oxygen. This means that
phytochemicals is affected by thermal processing as reported by (Davey et al., 2001 ).

4.4.2 Effect of processing conditions on total flavonoid content

4.4.2.1 Effect of blanching on TFC

The total flavonoid content in fresh Moringa oleifera was found to be 526.70 mg/g.
blanching decreased total flavonoid to 453.48 mg/g. The statistical analysis (One-way
ANOVA) showed that the flavonoid content was significantly decreased (p<0.05) during
blanching as compared to fresh moringa leaves.

The decreasing trend of flavonoid content during blanching is also reported by (Amin
and Lee, 2005). Blanching, a brief exposure of vegetables to high temperature alters the
constituents of plant tissue (Indrawati et al., 2000). Flavonoids are altered by changes in
temperature; however the thermal effect may vary with respect to the structural properties
of the compound and matrix of the tissue. The varying effect of blanching process
observed in the study was similar to that observed by (Oboh, 2005). Loss in
phytochemicals content due to blanching has been reported by (Gupta et al., 2012; Sikora
et al., 2008).

4.4.2.2 Effect of drying of TFC

During drying flavonoid content decreased from 526.7 mg/g to 377.4 mg/g. The statistical
analysis showed that there is significant difference in TFC of fresh and dry leaves. It was
also observed that TFC of moringa was significantly decreased during drying.

The percentage loss in flavonoid was observed to be 35.93% which was closer to the
observation made by (Zhang et al., 2009).Thus might be integrity of al structure is affected
due to the thermal destruction of flavonoids by various chemical reactions involving
enzymes, light and oxygen. This means that the phytochemical is affected by thermal
processing (Davey et al., 2001 ).

55
4.4.2.3 Effect of dry roasting on TFC

The total flavonoid content of fresh Moringa oleifera was found to be 526.7 mg/g. During
dry roasting of leaves, the flavonoid content decreased from 526.7 mg/g to 270 mg/g. The
statistical analysis (one-way ANOVA) showed that the TFC of moringa was significantly
decreased (p<0.05) during dry roasting. It was noted that, roasted moringa leaves showed
lower concentration on TFC than other processing methods.

Metabolism of phytochemical begins right after harvest and can involve complex
biochemical reactions. This reaction can lead to significant changes in plant attributes and
health promoting phytochemicals, such those with strong antioxidant activities (Hongyan
et al., 2012). Different phytochemicals are affected by temperature differently. Flavonoids
are sensitive to high heat and can incur significant losses during heat treatment. Ascorbic
acid is also a natural antioxidant which gets destroyed even at mild temperature (Hamauzu
and Zhang, 2004).

4.4.3 Effect of processing conditions on tannin content

4.4.3.1 Effect of blanching on tannin

The tannin content in fresh Moringa oleifera was found to be 12.81 mg/g and that after
blanching decreased to 8.36 mg/g. The statistical analysis (one-way ANOVA) showed that
tannin content was significantly decreased (p<0.05) during blanching. It is supported by a
result obtained by Makkar and Becker (1996), who found that moringa leaves are superior
in terms of tannin content.

Tannins are natural polyphenols distributed in plants such as vegetables, fruit and seeds
which are widely used in wine industry for color stabilizer, blanching the complexity in
wines, inhibit certain enzymes in infected fruits and acts as wine fining agents (Sanz et al.,
1997). It has also ability to precipitate proteins and alkaloids (Amarowicz, 2007).

4.4.3.2 Effect of drying on tannin

The tannin content present in the dried Moringa oleifera leaves were determined as per the
standard procedure provided. During drying tannin decreased from 12.81 mg/g to 9.64
mg/g. The observation for tannin in fresh moringa leaves is closer to the observation made
by (Makkar and Becker, 1996). It was noted that, dried leaves showed lower concentration

56
of tannins than of fresh leaves. Drying affects negatively phytochemicals was reported by
(Nobosse et al., 2017). Similar observation on decrease in tannin was reported by (Rakic et
al., 2007) in oak plant and the decrease in tannin content may be due to the result of
thermal degradation of hydrolysable tannin (Rakic et al., 2007).

4.4.3.3 Effect of dry roasting on tannin

The Tannin content of fresh Moringa oleifera leaves was found to be 12.81 mg/g. During
dry roasting of leaves, the tannins content decreased from 12.81 mg/g to 6.39 mg/g. The
statistical analysis (one-way ANOVA) showed that the tannin content of leaves was
significantly decreased (p<0.05) during dry roasting. It was showed that, roasted moringa
showed lower concentration of tannin than other methods of processing.

At low temperature, deterioration of phytochemicals are slowed down (Hongyan et al.,

2012) opposed to lower temperature, high temperature also bring a significant changes in
TPC, TFC, tannin content and antioxidant activity compared with its fresh form. However
their concentration may vary according to drying methods used and the duration exposure
to hot air (Michalczyk et al., 2009). Similarly, according to Reblova (2012) at high
temperature the TPC and antioxidant activity follows a decrease trend which is caused by
decrease in the ability of antioxidant to reacts with free radicals. The effect also coincides
in case of tannins as the hydrolysable tannins gets thermally degraded, causing an increase
in non-tannin content (Rakic et al., 2007).

4.4.4 Effect of processing conditions on chlorophyll content

4.4.4.1 Effect of blanching on chlorophyll

Chlorophyll content of fresh Moringa oleifera was found to be 23.27 mg/g and that after
blanching was found to be 17.32 mg/g. The statistical analysis (one-way ANOVA) showed
significantly difference (p<0.05) during blanching as compared to fresh leaves. Similar
results for chlorophyll content in leafy vegetables were obtained by (Nartnampong et al.,
2016).

The excessive heating of food products causes considerable losses in organoleptic

quality of food (Hayakawa and Timbers, 1977). Blanching inactivates chlorophyllase and
enzyme responsible for senescence and rapid loss of green color. Chlorophyll degradation
is initiated by damaged tissue during blanching and other processing steps (Heaton and
57
Marangoni, 1996; Tijekens et al., 2001). Hence low chlorophyll compared to fresh leaves
due to processing effects that the leaf has undergone.

4.4.4.2 Effect of drying on chlorophyll

Chlorophyll of dried Moringa oleifera leaves were determined as per the standard
procedure provided. During drying, chlorophyll content decreased from 23.27 mg/g to
17.28 mg/g. The statistical analysis (one-way ANOVA) showed significant different
(p<0.05) in chlorophyll content during drying. The observation for chlorophyll in dried
moringa oliefera is closer to that of observation made by (Nartnampong et al., 2016).
Abdulkadir et al. (2015) reported an average of 35.40±1.2 mg/g as high chlorophyll
content and average of 16.25±1.25 mg/g as low chlorophyll in fresh Moringa oleifera
leaves. The decrease in chlorophyll content in dried leaves due to the processing effects,
that leaf has undergone. Total chlorophyll decreased during drying. This indicates a high
potential for the acceptability of the dried leaves because they are desired by consumers in
the green state. Chop and dried vegetables retained substantial amounts of chlorophylls as
reported by (Adebooye and Singh, 2006).

4.4.4.3 Effect of dry roasting on chlorophyll

The chlorophyll content of fresh Moringa oleifera leaves was found to be 23.27 mg/g.
During dry roasting of leaves, chlorophyll content of leaves decreased from 23.27 mg/g to
6.39 mg/g. The statistical analysis (one-way ANOVA) showed that the chlorophyll content
of leaves was significantly decreased (p<0.05) during dry roasting. It was showed that,
roasted moringa showed lower concentration of chlorophyll than other methods of
processing.

4.4.5 Effect of processing conditions on antioxidant activity

4.4.5.1 Effect of blanching

Antioxidant activity of blanched moringa leaves were determined as per the standard
procedure provided. Antioxidant activity of fresh leaves was found to be 82.98% and that
after blanching was found to be 64.63%%. The statically analysis (one-way ANOVA)
showed that antioxidant activity significantly decreased (p<0.05) during blanching.

58
 Similar results were obtained by (Unuigbe et al., 2014) where antioxidant activity of
leaf in crude methanol was found to be 81.11%. Enhancement of antioxidant activity has
been reported in several leafy vegetables after blanching compared to raw samples
(Adefegha and Oboh, 2011; Ferracane et al., 2008). Loss of activity of some antioxidant
components during blanching might be a reason behind low antioxidant activities of the
blanched vegetables. Similar values of antioxidant activity for cruciferous vegetables were
obtained by (Amin and Lee, 2005).

4.4.5.2 Effect of drying

Antioxidant activity of dried moringa leaves were determined as per the standard
procedure provided. During drying, moringa leaves antioxidant activity decreases from
82.98% to 60.02%. Statistical analysis showed that antioxidant activity slightly decreased
(p<0.05) during drying.

Reduction is in accordance with the observation of (Wangcharoen and Gomolmanee,

2013) who recorded a 40% reduction in antioxidant activity of dried Moringa oleifera
leaves. Percentage loss in antioxidant activity of dried leaves was observed to be low when
compared to total phenol loss. This minimum loss in antioxidant activity could be due to
formation of mailard reaction product like hydroxymethyl furfural (HMF), which produces
high antioxidant activity (Duences, 2005; Siddhraju, 2005). The formation of mailard
reaction products could have masked the thermally destroyed polyphenol exhibiting
antioxidant activity. These complex chemicals interactions that influence the functional
properties of plant leaves during drying are still being researched.

4.4.5.3 Effect of dry roasting

Antioxidant activity of roasted Moringa oleifera was determined as per the standard
procedure provided. During roasting the antioxidant activity decreased from 82.98% to
52.73%. Statistical analysis (one-way ANOVA) showed that antioxidant activity
significantly decreased (p<0.05) during roasting. It was noted that, roasted moringa
showed lower concentration of antioxidant activity than other methods of processing.

Thermal drying processing methods have variable effects on antioxidant properties of

plant samples. Effect includes little or no changes, significant loss or enhancement in
antioxidant properties (Nicoli et al., 1999). In Moringa oleifera leaves thermal treatment

59
decreases the antioxidant activity. Many studies have been reported losses in antioxidant
activity of plant samples following thermal treatments. Losses were mainly reported in
Vegetables (Ismail et al., 2004; Roy et al., 2007; Toor and Savage, 2006). Losses in
antioxidant activity of heat treated samples have been attributed to thermal degradation of
phenolic compounds. Declines in antioxidant properties have been attributed to thermal
degradative enzymes, thermal degradation of phytochemicals and to loss of antioxidant
enzymes activities (Lim and Murtijaya, 2007). Declines in TPC and antioxidant activity are
often accompanied by loss of other bioactive properties (Roy et al., 2007)

4.5 Sensory evaluation of Moringa oleifera

Sensory evaluation was carried out for color, flavor, taste, mouth feel and overall
acceptability by semi trained panelists using 9 point hedonic scale. The statistical analysis
(two way ANOVA no blocking) was done. Post-Hoc test was carried out using LSD at 5%
level of significance. There was significant difference for most of the sensory attributes
viz., color, flavor, taste, mouth feel and overall acceptability at P<0.05. The result of the
sensory evaluation and statistical analysis is given in Table 4.5.

Table 4.5 Sensory evaluation of Moringa oleifera

Sample Color Flavor Taste After Taste Overall

Acceptability

A 6.33 (0.51)b 5.17 (0.40)b 5.17 (0.75)b 5.50 (0.55)a 5.83 (0.41)b

B 6.67 (0.81)bc 7.33 (0.52)c 8.00 (0.40)c 7.50 (0.55)b 7.50 (0.55)c

C 7.50 (0.54)c 7.67 (0.52)c 7.83 (0.54)c 7.00 (0)b 7.67 (0.52)c

D 4.16 (0.75)a 4.50 (0.84)a 4.50 (0.54)a 5.30 (0.89)a 4.50 (0.55)a

F pr. <.001 <.001 <.001 <.001 <.001

60
4.5.1 Color

The mean sensory score for the four samples of moringa A, B, C and D were found to be
6.33, 6.66, 7.5 and 4.16 respectively which is shown on Fig. 4.1. The highest score for the
color was obtained for sample C and least was obtained for sample D. Fresh sample was
significantly different (p<0.05) from different treatments. Similarly, the colored of
blanched, dried and roasted were also significantly different (p<0.05) from each other‟s.
LSD showed that A, B, C and D are significantly different from each other at 5% level of
significance.

A B C D
9
c
8
bc
b
7
Mean Sensory Value

5 a

0
A B C D
Treatment

Fig. 4.1 Mean sensory score for color

The values in the figure are the mean sensory score for color. Values on the top of bar
bearing similar alphabet are not significantly different at 5% level of significance. Vertical
error bar represent standard deviation of scores.

Color is a sensation that forms part of the sense of vision and judges the appearance of a
food product (Jellinek, 1985). Color is often used as an indicator to evaluate severity of the

61
heat treatment and to predict the corresponding quality degradation caused by blanching,
roasting process. The green color of leafy vegetables is desirable quality parameters by
consumers but processing interferes with color either positively or negatively depending on
the treatment (Margaret et al., 2017). In this case, processing has negatively affected color
of dry roasted leaves powder. Loss of color can be attributed to the loss in chlorophyll
because of leaching and chlorophyll forming pheophytin being broken down (Rodoni et
al., 1997). Processing of any food whether through drying or blanching is expected to
produce some changes in the sensory qualities of the product (Stanley et al., 1995).

4.5.2 Flavor

Mean sensory score for flavor sample A, B, C and D were found to be 5.16, 7.33, 7.66 and
4.5 respectively. The highest score for flavor was obtained for sample C and least was
obtained for sample D. So sample C were superior on the basis of flavor from statically
analysis which is shown in Fig. 4.2. The color of sample was significantly different
(p<0.05). LSD showed that sample A&B, A&C, A&D, B&D and C&D are significantly
different while other B&C are not significantly different from each other at 5% level of
significance.

A B C D
9
c c
8
7
Mean Sensory value

6 b
a
5
4
3
2
1
0
A B C D
Treatment

Fig. 4.2 Mean sensory score for flavor

62
The values in the figure are the mean sensory score for flavor. Values on the top of bar
bearing similar alphabet are not significantly different at 5% level of significance. Vertical
error bar represent standard deviation of scores.

Flavor includes taste and aroma perceived through tasting (Jellinek, 1985). Flavor is the
perception one gets after tasting food which includes both the taste and aroma of the food
(Stone and Sidel, 2004). Fresh leaves sample imparts leafy flavor, so it is less like
compared to blanching and dried samples. Dry Roasted imparts burny smoky flavor due to
high temperature treatments. Processing parameters (e.g. processing time, temperature ) is
responsible for changes the quality of food by altering the sensory attributes which
influences the consumer choices (Liyun, 2016).

4.5.3 Taste

The mean sensory score for Taste sample A, B, C and D were found to be 5.16, 8, 7.8 and
4.5 respectively. The highest score obtained for sample B and least obtained for sample D.
The color of the sample was significantly different (p<0.05) which is shown in Fig. 4.3 in
below. LSD showed that sample A&B, A&C, A&D, B&D and C&D are significantly
different while other B&C are not significantly different from each other at 5% level of
significance. Sample B were the superior on the basis of taste from statically analysis.

63
 A B C D
9 c c

7
b
Mean Sensory Value

6
a
5

0
A B C D
Treatment

Fig. 4.3 Mean sensory score for taste

The values in the figure are the mean sensory score for taste. Values on the top of bar
bearing similar alphabet are not significantly different at 5% level of significance. Vertical
error bar represent standard deviation of scores.

The sensation of taste is a result of the effect of water molecules interacting with
receptors on the tongue and in the oral cavity (Carpenter et al., 2000). Thermal processing
has impact on the positive and negative taste attributes of food stuffs (Liyun, 2016).
Samples B and C were found to be superior in terms of taste this might be the positive
impact of thermal processing of moringa leaves.

4.5.4 After-taste

The mean sensory score for flavor of four samples of moringa oliefera A, B, C and D were
found to be 5.5, 7.5, 7 and 5 respectively. The color of sample was significantly different
(P<0.05). The least score for after taste was obtained for sample D while the highest mean
score was obtained for sample B. The samples A&D and B&C were not found to be

64
significantly different while the other samples are found to be significantly different at 5%
level of significance which is shown in the Fig. 4.4. Sensory panelist preferred blanched
dried sample in context of aftertaste. After-taste is the lingering of the sense of taste of a
product on taste buds or the sensations perceived after food has been swallowed (Carpenter
et al., 2000).

A B C D
9
b
8 b

7
a
Mean Sensory value

6 a

0
A B C D
Treatment

Fig. 4.4 Mean sensory score for after-taste

The values in the figure are the mean sensory score for after taste. Values on the top of bar
bearing similar alphabet are not significantly different at 5% level of significance. Vertical
error bar represent standard deviation of scores.

4.5.5 Overall acceptability

The mean sensory score for overall acceptability of four samples A, B, C and D were
found to be 5.83, 7.5, 7.66 and 4.5 respectively. The color of sample was significantly
different (p<0.05). LSD showed that A&B, A&C, A&D and B&D are significantly
different while B and C are not significantly different from each other at 5% level of

65
significance. Sample B and C are superior on the basis of overall acceptability from
statically analysis which is shown in Fig. 4.5.

A B C D
9
c c
8

7
b
Mean Sensory Value

6
a
5

0
A B C D
Treatment

Fig. 4.5 Mean sensory score for overall acceptability

The values in the figure are the mean sensory score for overall acceptability. Values on the
top of bar bearing similar alphabet are not significantly different at 5% level of
significance. Vertical error bar represent standard deviation of scores.

Consumers are influenced differently by specific sensory attributes when they judge
overall acceptability (Moskowitz, 1994). Overall acceptability is the reflection of other
sensory attributes. On the basis of color, flavor, taste, sample B and C were most liked by
panelists. Statistically analysis showed higher acceptability for sample B and C.

4.6 Optimization study

As regard with the sensory scores and phytochemical analysis, the optimization study is
carried out as a given below.

66
Table 4.6 Optimized goals for treatment (Sensory)
Parameters Optimized Treatments

Color C

Flavor B=C

Taste B=C

After-taste B=C

Overall acceptability B=C

Table 4.7 Optimized goals for treatment (Phytochemical analysis)

Parameters Optimized treatment

TPC A

TFC A

Tannins D

DPPH radical scavenging activity A

Chlorophyll A

Regarding the parameters for phytochemical analysis treatment A, i.e., Fresh moringa leaf
was found to be superior but in terms of Sensory analysis treatment C, i.e., Cabinet dried
leaf was found to be superior.

67
 Part V

Conclusion and recommendations

5.1 Conclusion

Present work was carried out to study the effect of processing methods (water blanching,
cabinet drying and dry roasting) on the phytochemicals content and sensory attributes of
Moringa oleifera leaves. Based on this research following conclusion can be drawn.

 Proximate composition of fresh leaves were determined that is moisture, protein,

crude fiber, crude fat and ash content found to be 75.6%, 24.85%, 8.19%, 7.1%,
8.72% respectively.
 Proximate composition of powder leaves after treatments( water blanching, Cabinet
drying and dry roasting) were determined that is moisture, protein, crude fiber,
crude fat and ash content found to be (6.28%, 22.86%, 7.7%, 8.43%, 7.34%),
(5.95%, 27.67%, 9.40%, 8.23%, 7.55%) and (6.54%, 20.36%, 9.48%, 8.90%,
7.65%) respectively.
 Adequacy of blanching (Peroxidase/Catalase test) was found to be 98oC for 2 min.
 Blanching, drying and dry roasting of leaves significantly (p<0.05) decreased
phytochemicals content and had a significant effect of sensory attributes of
Moringa oleifera leaves
 It can be concluded that fresh leaves is superior to that of others in terms of
phytochemical analysis and dried leaves is superior in terms of sensory analysis. As
moringa leaves content good source of antioxidant so consumption of leaves either
in raw or cooked form can be the source of antioxidant, which is beneficial for
health.

5.2 Recommendations

The plant studied in this work is on high demand because of its high nutritional value and
traditional uses. Thus following suggestions are recommended for future work in the field
of Moringa oleifera leaves.

1. Antimicrobial activity of moringa leaves can be studied.

2. Preparation of different moringa leaves product and study its nutritional quality.

68
3. Storage stability of moringa powder can be studied.

69
 Part VI

Summary

Moringa oleifera leaves were collected in polythene bags from Tamor river banks of
Dhankuta District, Nepal for the study. Initially adequate time-temperature for blanching is
optimized and found to be 2 min at 98oC for water blanching, then subjected to blanching
and cabinet drying at 50oC and dry roasting 120oC. Proximate composition of fresh
Moringa oleifera leaves was analyzed. Crude extract of sample were prepared using 80%
methanol through maceration technique. Phytochemicals screening of the methanol extract
of the plant showed the presence of total phenol content, flavonoid, tannin and glycosides.
total phenol, flavonoid, tannin content, DPPH radical scavenging activity and chlorophyll
content for four samples: fresh sample, blanched dried sample, dried sample and dry
roasted sample were used for performing phytochemicals content, antioxidant activity and
chlorophyll content.

Drying, blanching and dry roasting decreased the levels of phytochemicals content and
antioxidant activity significantly (p≤0.05) as compared to fresh leaves. Drying slightly
effect chlorophyll content whereas blanching least affected chlorophyll content and dry
roasting most affect the chlorophyll content.

Fresh leaf is superior than others samples in terms of phytochemicals and antioxidant,
Blanched dried in terms of processing and cabinet dried leaves is superior in terms of
sensory analysis. This can be useful for the antimicrobial, nutritional requirements for the
daily life. This indicates that a bright scope for commercialization of fresh and dried
moringa oliefera leaves. It constitutes a large group of chemicals with high potential to
treat various diseases, so phytochemical analysis is the best way to bring forward Moringa
oleifera as medicinally important plant. Consumption of nutritionally and phytochemical
rich vegetables like moringa leaves should be promoted for greater consumption to
improve nutrition and strengthen immune functions for fighting infectious diseases.

70
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 Appendices
Appendix A

SPECIMEN CARD FOR SENSORY

Hedonic rating test

Name of the panelist ………………….

Date ………………………..

Name of the product: Moringa powder

Dear panelist, you have 4 samples of Moringa leaves. Please taste the sample and score
how much you prefer the each one. Please give point for your degree of preference for
each parameter as shown below using the scale given.

Parameter A B C D

Color

Flavor

Taste

After Taste

Overall acceptability

Give points as follows:

like extremely 9 like slightly 6 dislike moderately 3

like very much 8 neither like nor dislike 5 dislike very much 2

like moderately 7 dislike slightly 4 dislike extremely 1

Comment (if any)…………………………………………………………

Signature……………………

85
 Appendix B

Standard Curve of Gallic acid for phenol

1
y = 8.4333x - 0.0205
0.9 R² = 0.9902

0.8

0.7

0.6
Absorbance

0.5 abs

0.4 Linear (abs)

0.3

0.2

0.1

0
0 0.02 0.04 0.06 0.08 0.1 0.12
Concentration mg/ml

D.1 Gallic acid standard curve for phenol

86
Standard Curve of quercetin for flavonoid

0.012
y = 0.0958x - 0.0005
R² = 0.9749
0.01

0.008
Absorbance

0.006 abs
Linear (abs)

0.004

0.002

0
0 0.02 0.04 0.06 0.08 0.1 0.12
Concentration mg/ml

D.2 Quercetin standard curve for flavonoid

87
Standard Curve of Gallic acid for tannin

0.35 y = 3.0564x + 0.003

R² = 0.9482

0.3

0.25
Absorbance

0.2
abs
0.15 Linear (abs)

0.1

0.05

0
0 0.02 0.04 0.06 0.08 0.1 0.12
Concentration mg/ml

D.3 Gallic acid standard curve for tannin

88
 Appendix C

Table D.1 One way ANOVA (no blocking) for Phenol content

Source of d.f. s.s. m.s. v.r. F pr.

varaition

Sample 3 6804.62 2268.21 61.27 <.001

Residual 8 296.15 37.02

Total 11 7100.77

Table D.2 Least Significant differences of means (5%level) for Phenol content

Table Treatment

rep. 3

d.f 8

l.s.d 11.46

Table D.3 One way ANOVA (no blocking) for Flavonoid content

Source of d.f. s.s. m.s. v.r F pr.

variation

Sample 3 108442. 36147. 11.76 0.003

Residue 8 24598. 3075.

Total 11 133040.

89
Table D.4 Least Significant differences of means (5%level) for Flavonoid content

Table Treatment

Rep. 3

d.f 8

l.s.d 104.4

Table D.5 One way ANOVA (no blocking) for Tannin content

Source of d.f. s.s. m.s. v.r F pr.

varaition

Sample 3 65.5494 21.8498 90.98 <0.01

Residual 8 1.9214 0.2402

Total 11 67.4707

Table D.6 Least Significant differences of means (5%level) for Tannin content

Table Treatment

Rep. 3

d.f 8

l.s.d 0.923

90
Table D.7 One way ANOVA (no blocking) for Antioxidant activity

Source of d.f s.s. m.s. v.r F pr.

variation

Sample 3 1496.9078 498.9693 848.03 <.001

Residual 8 4.7071 0.5884

Total 11 1501.6194

Table D.8 Least Significant differences of means (5%level) for Antioxidant activity

Table Treatment

Rep. 3

d.f 8

l.s.d 1.444

Table D.9 One way ANOVA (no blocking) for Chlorophyll

Source of d.f s.s. m.s. v.r. F pr.

variation

Sample 3 402.7196 134.2399 254.86 <.001

Residual 8 4.2138 0.5267

Total 11 406.9334

91
Table D.10 least Significant differences of means (5%level) for Chlorophyll

Table Treatment

Rep. 3

d.f 8

l.s.d 1.366

Table D.11 Two way ANOVA (no blocking) for Color

Source of d.f. s.s. m.s. v.r. F pr.

varaition

Sample 3 36.3333 12.1111 25.35 <.001

Panelists 5 1.8333 0.3667 0.77 0.587

Residual 15 7.1667 0.4778

Total 23 45.3333

Table D.12 least Significant differences of means (5%level) for Color

Table Sample Panelists

Rep. 6 4

d.f. 15 15

l.s.d 0.851 1.042

92
Table D.13 Two way ANOVA (no blocking) for Flavor

Source of d.f. s.s. m.s. v.r. F pr.

variation

Sample 3 44.3333 14.7778 53.20 <.001

Panelists 5 2.8333 0.5667 2.04 0.131

Residual 15 4.1667 0.2778

Total 23 51.3333

Table D.14 least Significant differences of means (5%level) for Flavor

Table sample Panelists

Rep. 6 4

d.f. 15 15

l.s.d 0.649 0.794

Table D.15 Two way ANOVA (no blocking) for Taste

Source of d.f. s.s. m.s. v.r. F pr.

variation

Sample 3 58.4583 19.4861 77.09 <.001

Panelists 5 1.3750 0.2750 1.09 0.407

Residual 15 3.7917 0.2528

Total 23 63.6250

93
Table D.16 least Significant differences of means (5%level) for Taste

Table Sample Panelists

Rep. 6 4

d.f. 15 15

l.s.d. 0.619 0.758

Table D.17 Two way ANOVA (no blocking) for After taste

Source of d.f. s.s. m.s. V.r. F pr.

variation

Sample 3 25.5000 8.5000 23.18 <.001

Panelists 5 1.5000 0.3000 0.82 0.555

Residual 15 5.5000 0.3667

Total 23 32.5000

Table D.18 least Significant differences of means (5%level) for After Taste

Table sample Panelists

Rep. 6 4

d.f. 15 15

l.s.d 0.745 0.913

94
Table D.18 Two way ANOVA (no blocking) for Overall acceptability

Source of d.f. s.s. m.s. v.r. F pr.

variation

Sample 3 40.4583 13.4861 47.14 <.001

Panelists 5 0.8750 0.1750 0.61 0.693

Residual 15 4.2917 0.2861

Total 23 45.6250

Table D.19 least Significant differences of means (5%level) for Overall acceptability

Table sample Panelists

Rep. 6 4

d.f. 15 15

l.s.d. 0.658 0.806

95
 List of Plates

P1 Fresh moringa leaves

P2 Extract preparation

96
 P3 Sensory analysis

P4 Phytochemical analysis

97