MICROENCAPSULATED NUTRACEUTICAL PRODUCTS

DEVELOPMENT AND MEDICINAL VALUES OF MORINGA

STENOPETALA LEAVES EXTRACT

A Dissertation

By

DEBEBE WORKU DADI

Submitted to the School of Chemical and Bio-Engineering in Partial Fulfillment of

the Requirement for the Degree of

DOCTOR OF PHILOSOPHY (FOOD ENGINEERING STREAM)

Advisors: Dr. Eng. Shimelis Admassu Emire (Associate Professor)

Dr. Asfaw Debella Hagos (Lead Researcher)

Addis Ababa Institute of Technology, Addis Ababa University

Addis Ababa, Ethiopia

June, 2019
 0

MICROENCAPSULATED NUTRACEUTICAL PRODUCTS

DEVELOPMENT AND MEDICINAL VALUES OF MORINGA

STENOPETALA LEAVES EXTRACT

A Dissertation

By

DEBEBE WORKU DADI

Submitted to the School of Chemical and Bio-Engineering in Partial Fulfillment of

the Requirement for the Degree of

DOCTOR OF PHILOSOPHY (FOOD ENGINEERING STREAM)

Advisors: Dr. Eng. Shimelis Admassu Emire (Associate Professor)

Dr. Asfaw Debella Hagos (Lead Researcher)

Addis Ababa Institute of Technology, Addis Ababa University

Addis Ababa, Ethiopia

 June, 2019

0
Author’s Declaration

This dissertation contains no material which has been accepted for the award of any other degree

or diploma in any university or other tertiary institution and, to the best of my knowledge and

belief, contains no material previously published or written by other persons, except where due

reference has been made in the text.

Debebe Worku Dadi

Signature: ______________
 i
Abstract

Bioactive compounds are secondary metabolites of the plant, which are produced to increase

their overall ability to survive and withstand the harsh environment and resists to diseases.

Moringa stenopetala plant contains bioactive compounds, which have antihypertensive, anti

inflammatory, antihyperglycemic and prevention of cancer. However, during food processing

such as thermal processing, these bioactive compounds can to be destructed. Hence,

encapsulation might be an alternative technology to maintain the functionality of the active

compounds. Therefore, the aim of this research was to develop microencapsulated nutraceutical

product from M. stenopetala leaves extract. The microencapsulation was done using spray and

freeze-drying encapsulation techniques. In addition, maltodextrin (MD) and mixtures of

maltodextrin and high methoxyl pectin (MDHP) were used as coating materials. Then, the

physical and functional properties, encapsulation efficiency, bioactive contents and antioxidant

activities of the microencapsulates were measured. Moreover, the storage stability and in vitro

digestibility of the microencapsulated product was determined. Finally, the medicinal values of

the microencapsulated bioactive product were also evaluated using animal experiments.

According to the results, the spray- and freeze-drying encapsulation techniques, and coating

materials showed significant (P < 0.05) differences in the physical and functional properties, total

phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of the

microencapsulates. The encapsulation efficiency (EE) of spray dried microencapsulate was

significantly more (83.52–87.93%) than freeze-dried microencapsulate (71.44–82.12%).

Moreover, the EE was also significantly affected when the coating material was MDHP (87.93%)

compared to MD (83.52%). On the other hand, the TPC, TFC and antioxidant activities of the

freeze-dried microencapsulate were significantly (P < 0.05) higher when it was compared

i
to the spray dried microencapsulate. Moreover, TPC, TFC and the antioxidant activities of the

microencapsulates coated with MDHP were significantly more than the MD coated product. The

storage stability of the spray dried microencapsulate were better than the freeze-dried

microencapsulate. This might be due to the higher EE of the spray dried microencapsulate which

in turn the destruction of the bioactive compounds of freeze-dried microencapsulate would be

more during storage. Regarding the in vitro digestibility of the microencapsulate, the release of
TPC (73.08–75.20 mg GAE/g dm) and TFC (34.72–36.19 mg CE/g dm) from the freeze dried

microencapsulated product were more when it was compared to the release of TPC and TFC

from the spray dried microencapsulate, which were 63.49–70.42 mg CE/g dm and 26.08–33.81

mg CE/g dm, respectively. This probably is related to the lower EE of the freeze-drying process

and the more porous structure of the freeze-dried materials. On the other hand, the digestibility

of the microencapsulate coated with MDHP was significantly (P < 0.05) higher in simulated

intestinal fluid (SIF) compared to the simulated gastric fluid (SGF). On the contrary, the higher

digestibility of the microencapsulate coated with MD was founded in SGF. Although the TPC,

TFC and antioxidant activities of the freeze-dried microencapsulate were higher, the EE and

storage stability were lower when it was compared to the spray dried microencapsulate.

Therefore, spray drying microencapsulation process using MDHP as coating material can be an

alternative method to develop microencapsulated nutraceutical product from M. stenopetala

leaves extract. When the medicinal values of the microencapsulate product are concerned, there

were no toxicity signs were observed on the animals’ behavior, body weight and growth

pathology up to the dose of 5000 mg/kg of the body weight and on the control groups. In

addition, the animal experiments showed that the microencapsulated bioactive product of M.

stenopetala leaves extract had antihyperglycemic, vasodilator (up to 74.17% of relaxation) and

ii
diuretic activities (up to 56% of urinary excretion). Therefore, it can be concluded that the

microencapsulation technologies use for the development of nutraceutical product from natural

resources for global marketing values. However, further studies are recommended for chronic

toxicity test and human trial for the medicinal values of the microencapsulated bioactive product.

Keywords: Moringa stenopetala, extraction, microencapsulation, microencapsulation

techniques, quality analysis, medicinal values

iii
Acknowledgments

I wish to express my earnest appreciation to my supervisor Dr. Eng. Shimelis Admassu for his

continuous moral, follow up, wisdom and knowledge that were critical to my success as a

graduate student. He helped me to overcome the difficulties and the completion of my study
successfully.

I would also like to extend my appreciation towards my co-supervisor Dr. Asfaw Debella Hagos,

a lead researcher at Ethiopian Public Health Institute, Ethiopia, for his continuous guidance,

critical thinking, especially on laboratory techniques and encouragement.

It is a pleasure to thank Professor Jong-Bang Eun, at Chonnam National University, South Korea,

for his willingness to use his laboratory facilities, valuable time and guidance.

I would like to thank Addis Ababa University, Ambo University, Chonnam National University

and National Institute for International Education for granting me a scholarship and Ethiopian

Public Health Institute for laboratory facilities that allowed me to finish this PhD research work

My thanks go to Professor Kim-Young Soo, for his willingness to use the spray dryer and Mrs.

Hae Mi Kim for her technical support to operate the spray dryer, Chonbuk National University,

South Korea.

I would like to thank Arba Minch University for the gift of the sample from the farm and to use

the laboratory for sample treatment and drying.

I would like to thank my friends and staffs at Addis Ababa Institute of Technology, Ambo

University, Ethiopian Public Health Institute, and Department of Food Science and Technology,

iv
Chonnam National University, Korea, for their moral support throughout my PhD studies. A

deepest appreciation is extended to all my friends for your continuous motivating and

encouraging words that made me successful.

Finally yet importantly, I would like to express my deepest gratitude to my families. This

dissertation would not have been possible without their warmest love, continued patience and

endless support. Thanks to my wife, Meron Hailessilassie for her constant love, support and

encouragement have sustained me throughout my work and life.

v
List of publications

Dadi Debebe W., Shimelis A. Emire, Asfaw D. Hagos, and Frehiwot T. Assamo. Influences of
 Different Drying Methods and Extraction Solvents on Total Phenolic and Flavonoids, and

Antioxidant Capacity of Moringa stenopetala Leaves. Journal of Pharmacognosy and

Phytochemistry 7 (2018): 962-967.

Dadi Debebe W., Shimelis A. Emire, Asfaw D. Hagos, and Eun, J.B. Improvement of Yield of

Bioactive Compounds and Antioxidant Activity of Moringa stenopetala Leaves by

Optimizing Ultrasonic-assisted Extraction. Research and Reviews: Journal of Food Science

and Technology 7 (2018):1-11.

Dadi Debebe W., Shimelis A. Emire, Asfaw D. Hagos, and Eun, J.B. Evaluation of Ultrasonic

Assisted Extraction of Moringa stenopetala Leaves on Bioactive Compounds and Antioxidant

Effect. Journal of Food Technology and Biotechnology 57 (2019): 77-86.

Dadi Debebe W., Shimelis A. Emire, Asfaw D. Hagos, and Eun, J.B. Development of

Microencapsulated Bioactive Product from Moringa stenopetala Leaves Extract by

Optimizing Spray Drying Process Parameters. Annals. Food Science and Technology 20

(2019). (Accepted).

vi
Table of Contents
 Title Page

Abstract i Acknowledgments iv Table of Contents vii List of Figures xiii List of Tables xiv

List of Abbreviations xvii Chapter 1 Introduction 1

1.1 Background 1 1.2 Statement of the problem 2 1.3 Hypothesis 4 1.4 Research questions 4 1.5
General objective 4 1.6 Specific objectives 5 1.7 Organization of the dissertation 5 Chapter 2
Literature Review 7

2.1 General overview of Moringa stenopetala plant 7 2.2 Nutritional composition of Moringa
plant 8 2.3 Bioactive compounds 9 2.4 Extraction techniques of bioactive compounds 11 2.5
Encapsulation for food industry 12 2.6 Benefits of encapsulation in food processing 14 2.7
Selection of encapsulating materials for food industry 15 2.8 Encapsulation technologies 17
2.9 Concluding remarks 19

vii
Chapter 3 General Materials and Methods 21

3.1 Sample collection, transportation and preparation 21 3.2 Chemicals and reagents 21 3.3
Moisture content determination 22 3.4 Extraction Methods 22 3.4.1 Convectional (Maceration)
extraction 22

3.4.2 Ultrasonic-assisted extraction 23

3.5 Quantitative analysis of bioactive compounds and antioxidant activities of the extracts 23
3.5.1 Determination of total phenolic contents 23

3.5.2 Determination of total flavonoid contents 24 3.5.3 DPPH radical scavenging activity assay 25

3.5.4 ABTS radical scavenging assay 25 3.5.5 Ferric reducing power assay 26 3.5.6 Ferrous ion

chelating activity assay 26

3.6 Experimental design and statistical data analysis 27 Chapter 4 Effects of Drying
Methods and Extraction Solvents on Total Phenolic and

Flavonoids Content, and Antioxidant Capacity of Moringa stenopetala Leaves 28

 Abstract 28 4.1 Introduction 29 4.2 Materials and methods 31 4.3 Results and discussion 33
4.3.1 Effect of drying methods on residual moisture content dried sample 33

4.3.2 Effect of drying methods and extraction solvents on the extraction yield 34 4.3.3 Total phenolic

contents 36 4.3.4 Total flavonoid content 38 4.3.5 DPPH radical assay 41

viii
4.3.6 ABTS radical assay 43

4.4 Conclusions 45 Chapter 5 Improving the Yield of Bioactive Compounds and

Antioxidant Activities

of Moringa stenopetala Leaves by Optimizing Ultrasonic-Assisted Extraction 46

Abstract 46 5.1 Introduction 47 5.2 Materials and methods 48 5.3 Results and discussion 50
5.3.1 Fitting response surface models 50

5.3.2 Influence of UAE parameters on total phenolic content 52 5.3.3 Influence of UAE parameters

on total flavonoid content 56 5.3.4 Influence of UAE parameters on DPPH and ABTS radical

scavenging activity 58 5.3.5 Optimization of UAE parameters and model validation 61 5.3.6

Comparison of UAE and maceration 62

5.4 Conclusions 63 Chapter 6 Effects of Ultrasonic Temperature and Time of Extraction

of Moringa

stenopetala Leaves on Bioactive Compounds and Antioxidant Activity 64

Abstract 64 6.1 Introduction 65 6.2 Materials and methods 67 6.3 Results and discussion 70
6.3.1 Effects of UAE temperature and time on TPC and TFC 70

6.3.2 Effect of UAE temperature and time on the antioxidant activities 73 6.3.3 Bound TPC and TFC

75 6.3.4 Antioxidant activities of bound phenolic extracts 77 6.3.5 Morphological analysis 79

ix
6.4 Conclusions 81 Chapter 7 Encapsulation Efficiency, Digestibility, and Storage
Stability of Spray
Dried and Freeze-Dried Microencapsulated Bioactive Products from Moringa stenopetala

Leaves Extract 82

Abstract 82 7.1 Introduction 83 7.2 Materials and methods 85 7.3 Results and discussion 94
7.3.1 Moisture content 94

7.3.2 Bulk and tapped density 95 7.3.3 Flowability 96 7.3.4 WAI and WSC 96 7.3.5 Hygroscopicity

98 7.3.6 Dispersibility 99 7.3.7 Color 99 7.3.8 Particle size distribution 101 7.3.9 Encapsulation

efficiency 103 7.3.10 TPC and TFC microencapsulated product 103 7.3.11 Antioxidant activities

microencapsulated product 105 7.3.12 In vitro digestibility of microencapsulated nutraceutical

products 105 7.3.13 Storage stability 107 7.3.14 Morphology of microencapsulated nutraceutical

products 110

7.4 Conclusions 112 Chapter 8 Optimization of Spray Drying Process Conditions to

Microencapsulate

the Bioactive Compounds from Moringa stenopetala Leaves Extract 113

x
Abstract 113 8.1 Introduction 114 8.2 Materials and Methods 116 8.3 Results and discussion
121 8.3.1 Analysis of extract before encapsulation 121

8.3.2 Fitting the response surface model 121 8.3.3 Moisture content and water activity 124 8.3.4

Bulk and tapped density 126 8.3.5 Water absorption index 128 8.3.6 Water solubility capacity 129

8.3.7 Hygroscopicity 130 8.3.8 Dispersibility 131 8.3.9 Flowability 133 8.3.10 Encapsulation

efficiency of microencapsulated product 134 8.3.11 The TPC and TFC content of microencapsulated

product 137 8.3.12 Antioxidant activity of microencapsulated bioactive product 141 8.3.13 Process

optimization of spray drying 142 8.3.14 Color 142 8.3.15 Particle size distribution 145 8.3.16

Evaluation of the digestibility microencapsulated product 147 8.3.17 Multivariate analysis 148

8.3.18 Morphology of microencapsulates 150 8.3.19 Conclusions 152

Chapter 9 Evaluation of the Medicinal Values of Microencapsulated Nutraceutical

Product from Moringa stenopetala Leaves Extract 153

xi
Abstract 153 9.1 Introduction 154 9.2 Materials and methods 155 9.3 Results and discussion
161 9.3.1 Acute toxicity 161

9.3.2 Antihyperglycemic activities 161 9.3.3 Vasodilator activity 165 9.3.4 Diuretic activity 166

9.3.5 Conclusions 172

Chapter 10 General Conclusions and Recommendations 173

10.1 General conclusions 173 10.2 Recommendations 175 References 176

Appendix A Qualitative Analysis of Bioctive Compounds in the Extracts using Thin Layer

Chromatography 198 Appendix B Standard Curves for Bioactive Compounds Quantification 199

Appendix C Standard Curves for Antioxidant Activities 200 Appendix D Inlet Air Temperature

of Spray Dryer 201 Appendix E Key Procedures for Microencapsulated Product Development

202

xii
List of Figures

Figure # Title Page

 5.1: Response surface for the combined effects on the total phenolic content of the extract from M.

stenopetala extracts 55 5.2: Response surface for the combined effects on the total flavonoid content

of the extract from M. stenopetala extracts 57 5.3: Response surface for the combined effects on

DPPH from M. stenopetala extracts 59 5.4: Response surface for the combined effects on ABTS

from M. stenopetala extracts 60 6.1: Mass fractions of bound total phenolic and flavonoid contents

on dry mass basis of Moringa stenopetala leaves at different extraction time 76 6.2: The

antioxidant activity expressed on bound phenolic compounds extracted from Moringa stenopetala

leaves by ultrasonic-assisted extraction at different extraction time 78 6.3: The morphological

images of the sample after ultrasonic-assisted extraction 80 7.1: Digestibility of the

microencapsulated bioactive products from M. stenopetala leaves extract in terms of total phenolic

content and total flavonoid content 106 7.2: Storage stability of microencapsulated products from

M. stenopetala leaves extract 108 7.3: Morphological structures of microencapsulates using

scanning electron microscopy 111 8.1: Response surface for the combined effects on the

encapsulation efficiency of the microencapsulates 136 8.2: Response surface for the combined

effects on the total phenolic content of the microencapsulated product from M. stenopetala leaves

extract 138 8.3: Morphology of the microencapsulates developed at different spray drying

conditions. 151

xiii
List of Tables

Table # Title Page

4.1. Effects of different drying methods on the moisture content of M. stenopetala leaves. 34 4.2.
Effect of drying methods and extraction solvents on the extract yield from M. stenopetala leaves. 35

4.3 Effects of drying methods and extraction solvents on total phenolic contents of M. stenopetala

extracts 37 4.4. Effects of drying methods and extraction solvents total flavonoid contents of M.

stenopetala extracts 39 4.5. Effects of different drying methods and extracting solvents on DPPH

radical scavenging activities of M. stenopetala leaves extracts 42 4.6. Effect of different drying

methods and extracting solvent on ABTS radical scavenging activities of M. stenopetala leaves

extracts 44 5.1. Experimental design with the observed responses of TPC, TFC, DPPH, and ABTS

radical scavenging capacity of the extracts 51 5.2. Regression coefficients for the fitted quadratic

polynomial model and analysis of variance for the experimental results of TPC, TFC, IC 50 values of

DPPH, and ABTS radicals 53 6.1. Total phenolic and flavonoid contents on dry mass basis of

Moringa stenopetala leaves extracted by ultrasonication at different temperatures and time, and by

maceration 71 6.2. Antioxidant activity of Moringa stenopetala leaves extracted by ultrasonication

at different temperatures and time, and by maceration 74

xiv
7.1. Physical and functional properties of the microencapsulated bioactive products from M.

stenopetala leaves extract 97 7.2. Effect of spray- and freeze-drying techniques and coating

materials on the color of microencapsulated bioactive products from M. stenopetala leaves extract

100 7.3. Particle size distribution of the microencapsulated bioactive product from M. stenopetala

leaves extract 102 7.4. Encapsulation efficiency, bioactive content, and antioxidant activities of the

microencapsulated bioactive products from M. stenopetala leaves extract 104 8.1. Experimental

design with the observed responses of encapsulation efficiency and TPC of the microencapsulated

bioactive compound from M. stenopetala leaves extract 122 8.2. Regression coefficients for the
 fitted quadratic polynomial model and analysis of variance for the experimental results of

encapsulation efficiency and TPC 123 8.3. Moisture content, water activity and density of the

microencapsulated bioactive product of M. stenopetala extract 127 8.4. Water absorbance and

solubility capacity, hygrscopicity and dispersiblity of the spray dried microencapsulated bioactive

product 130 8.5. Evaluation of the flowability of the microencapsulates using Hausner’s ratio, Carr’s

index and angle of repose 132 8.6. Surface phenolic content, total flavonoid content and antioxidant

activities of the microencapsulated bioactive product from M. stenopetala leaves extract 140

8.7.Color values of the spray dried microencapsulated bioactive product of M. stenopetala leaves

extract 144

xv
8.8. Particle size distribution of the microencapsulated bioactive product from M. stenopetala leaves

extract 146 8.9. Evaluation of the digestibility of microencapsulated TPC in simulated gastric fluid

and intestinal fluid 148 8.10. Correlation analysis of the microencapsulated bioactive products’

variables 166 9.1. Effects of the microencapsulated bioactive product of M. stenopetala on the body

weight of the mice 162 9.2. Antihyperglycemic effects of microencapsulated bioactive product of

M. stenopetala leaves extract. 164 9.3. Vasodilation effect of the microencapsulated bioactive

product from M. stenopetala leaves extract on the guinea pig thoracic aorta. 165 9.4. Urine output

from the rats treated with microencapsulated bioactive product of M. stenopetala leaves extract.

167 9.5. Diuretic activity of the rat treated by the microencapsulated bioactive product 168 9.6.

Electrolyte extraction in the treated rats using the microencapsulated bioactive product. 171
 xvi
List of Abbreviations

BB Box-Behnken

ABTS 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt AMPK

Activation of protein kintase

ANOVA Analysis of variance

AOAC Association of analytical chemistry

CCRD Central composite rotatable design

CE Catechin equivalent

DPPH 2, 2-diphenyl-1-picrylhydrazyl

EDTA Ethylenediaminetetraacetic acid

FD Freeze dryer

FRAP Ferrous reducing antioxidant power

GAE Gallic acid equivalent

H Height
M Molar

MD Maltodextrin

MDHP Mixture of maltodextrin and high methoxyl pectin

mM Millimolar

nm Nanometer

RE Rutin equivalent

RSM Response surface methodology

SD Spray dryer

xvii
SGF Simulated gastric fluid

SIF Simulated intestinal fluid

SPC Surface phenolic content

TE Trolox equivalent

TPC Total phenolic content

TFC Total flavonoid content

TPTZ 2, 4, 6-Tris (2-pyridyl)-s-triazine

Trolox 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid UAE

Ultrasonic-assisted extraction

WAI Water absorbance index

WSC Water solubility capacity

 xviii
Chapter 1

Introduction

1.1 Background

Bioactive compounds are secondary metabolites of the plant, which are produced to increase

their overall ability to survive and withstand challenges by allowing them to interact with their

surroundings (Bernhoft, 2010; Harbone, 1993). The importance of the antioxidant properties of

some of these bioactive compounds and their possible use in processed foods as a natural

antioxidant has been increasing (Shi, 2006).

Some of the most common bioactive compounds include glucosinolates, flavonoids, tocopherol,

lecithin, ascorbic acid, citric acid, polyphenols, and others (Shi, 2006). Phenols and flavonoids

are major bioactive constituents of Moringa leaves extracts and have been reported to exhibit

antioxidant activity both in vitro and in vivo (Chumark et al., 2008; Vongsak et al., 2013a).

Bioactive compounds from plant origin like polyphenol compounds interfere with the formation

of free radicals that prevent the formation of hydroperoxides. However, during food processing,

especially conventional thermal processing (Shi, 2006) and storage at high temperature and

relative humidity (Vongsak et al., 2013b), these bioactive components are being significantly

reduced.

Utilization of the indigenous plant, M. stenopetala, for the production of bioactive compounds is

important to bring about for industrialization and potential utilization. In Ethiopia, there is high

cultivation of Moringa trees and availability of promising areas to expand the production, which

in turn increase the biosynthesis of bioactive compounds. Therefore, to maximize the utilization

1
of this plant, microencapsulation of the bioactive compounds might be important to develop

nutraceutical products.

Encapsulation is applied to food supplements to prevent the release of the active agent before it

reaches the specific area in the gastrointestinal tract and minimizes the destruction of the

bioactive compounds not to be broken down by the acidic environment of the digestive system

(Zuidam and Shimoni, 2010). Therefore, encapsulation is required to deliver bioactive

compounds to the appropriate site where it can be readily absorbed; besides, it masks the bitter

taste and odor of the product (Zuidam and Shimoni, 2010). On the other hand, bioactive
compounds present in Moringa plant are highly susceptible to high temperature, light, oxygen

and other environmental factors. The investigation of bioactive stability of the leaves extract from

Moringa has been limited. Therefore, encapsulation might be an alternative technology to

improve the stability and minimize the destruction of the bioactive compounds during

processing. The nutritional and the pharmaceutical properties of the plants were reported.

However, no research has been reported on the development of microencapsulated nutraceutical

products from M. stenopetala plant extract.

1.2 Statement of the problem

There is high production and potential areas to increase the cultivation of this plant. As described

by Abuye et al. (2003), the community discards the water that was used for boiling the leaves to

minimize the bitter taste, hence the loss of essential nutrients and bioactive compounds are

removed as well.

2
During processing, in particular, thermal treatments can lead to high losses of the bioactive

compounds. The bioactive compounds of moringa leaves extracts were lost during storage,

especially at high temperature and relative humidity (Vongsak et al., 2013b), which decreases the

nutritional and health benefits of the plant. According to the findings of Arun et al. (2011), it has

been estimated that about one-fourth of the harvested Moringa oleifera leaves were spoiled

before consumption. Moreover, 40% of the antioxidant activity of the leaves was lost during hot

air drying of the leaves (Wangcharoen and Gomolmanee, 2013). Different processing methods

have significant effects on the reduction of vitamin C due to leaching and thermal breakdown
(Davey et al., 2000). Therefore, many processes have effects on the total phenolic and flavonoid

compounds that in turn affect the health related quality of the plant.

On the other hand, increasing awareness and demand for natural bioactive compounds by the

consumer, strengthen the research in this area (Shi, 2006), since the availability of such organic

bioactive compounds is rather limited. Consequently, extraction of bioactive compounds from M.

stenopetala leaves and the development of natural bioactive products that can be used as food

additives to substitute synthetic antioxidants, often linked to carcinogenic effects is highly

desirable (Clevidence et al., 1997). Therefore, to maximize the utilization and industrialization of

this underutilized plant, the development of microencapsulated nutraceutical products can be an

alternative technology, which could create global market of natural extracts and supplements as

well as maximize the utilization of plant resources and thereby create new employment

opportunities.

3
1.3 Hypothesis

Microencapsulation can improve the quality, control release and storage stability of the bioactive

compounds extracted from M. stenopetala leaves.

1.4 Research questions

∙ Do different drying methods and extraction solvent have significant effects on the yield of
 bioactive compounds and antioxidant activities?

∙ Are the bioactive contents and antioxidant activities affected by ultrasonic assisted

extraction parameters?

∙ Does the spray drying and freeze-drying microencapsulation technologies have considerable

effects on the quality of the microencapsulated bioactive product? ∙ Do the spray drying

process parameters affect the quality of the microencapsulated product?

∙ Does the microencapsulated product have improved storage stability for the total phenolic

content of the extract?

∙ Does microencapsulates have medicinal values as the antihyperglycemic effects, vasodilator

and diuretic activities?

1.5 General objective

The overall objective of this study is to develop microencapsulated nutraceutical products from

M. stenopetala leaves extract and evaluate the medicinal values of the microencapsulated

products.

4
1.6 Specific objectives

The specific objectives were:

∙ Evaluate the influences of different drying methods and extraction solvents on the bioactive

compounds and antioxidant activities.

∙ Evaluate the effects of temperature and time of ultrasound-assisted extraction on the yield of

bioactive compounds and antioxidant activities from M. stenopetala leaves. ∙ Optimize the
 ultrasonic-assisted extraction method to improve the yield of bioactive compounds and

antioxidant activities of M. stenopetala leaves extract.

∙ Study the influence of spray drying and freeze-drying microencapsulation techniques on the

encapsulation efficiency, bioactive compounds, digestibility and storage stability of the

product from M. stenopetala leaves extract.

∙ Optimize spray drying microencapsulation process conditions to improve the encapsulation

efficiency and bioactive content of microencapsulated bioactive product from M.

stenopetala leaves extract.

∙ Assess the medicinal potential of the microencapsulated bioactive products using animal

experiments on antihyperglycemic, vasodilator and diuretic activities.

1.7 Organization of the dissertation

This dissertation comprises of ten chapters:

Chapter 1 provides the background of the study, a brief overview of the problem, the objective of

the study and the potential contributions of the findings of food and pharmaceutical industries.

Research hypothesis, statements of the problem, research questions, overall and specific

objectives are also included in this chapter.

5
Chapter 2 reviews current and past issues related to M. stenopetala plant, bioactive compounds,

extraction methods, microencapsulation techniques, influence of spray drying processes,

optimization techniques and coating material properties.

Chapter 3 provides different research material preparations and storage as well as general

analytical and statistical methods.

Chapter 4 describes the study of the influences of different drying methods and extraction

solvents on the bioactive contents and antioxidant activities of M.stenopetala leaves. Chapter 5

describes the study on the improvement of the yield of bioactive compounds and antioxidant

activities from M. stenopetala by optimizing ultrasonic-assisted extraction. Chapter 6 presents

the study of the effects on ultrasonic temperature and time of extraction of M. stenopetala leaves

on bioactive compounds and antioxidant.

Chapter 7 describes a study on the effects on spray drying and freeze drying microencapsulation

techniques using different coating materials on the functional properties, bioactive content,

digestibility and storage stability of the microencapsulates.

Chapter 8 describes the study on the optimization of spray drying microencapsulation process

conditions on the encapsulation efficiency and total phenolic contents, the physicochemical

properties of the microencapsulates with the parameters: core to coating ratio, maltodextrin to

pectin ratio and inlet air temperature.

Chapter 9 describes the study on the medicinal values: antihyperglycemic, vasodilator, and

diuretic activities of the microencapsulates using animal experiments.

Chapter 10 is the final chapter that provides the general conclusion, contribution to science and

knowledge, and recommendations made from the present research findings.

6
Chapter 2

Literature Review

2.1 General overview of Moringa stenopetala plant

Moringaceae, a monogeneric family with a single genus Moringa, which is characterized by 13

species of dicotyledonous tropical and sub-tropical flowering trees (Jahn, 1991; Jiru et al., 2006).

Although most Moringa species originated in India and Africa, now it is distributed in different

tropical countries (Amaglo et al., 2010). M. oleifera is native to Sub-Himalayan part of northern

India, commonly called as “horseradish tree” or “drumstick tree”, which has gotten great

emphasis on research. On the other hand, M. stenopetala, often referred to as the African

Moringa tree, is a multipurpose tree native to Ethiopia, northern Kenya and eastern Somalia,

which has a wide range of adaptation from the arid to humid climates (Jahn, 1991).

Moringa stenopetala is locally called Shiferaw in Amharic or Haleko in Gamo and Wolyeta, and

cabbage tree in English (Abuye et al., 2003). The botanical description of this tree: the height is

6–12 m with a diameter of 60 cm, a smooth bark, strongly branched, sometimes with several

trunks, and its wood is soft (Abuye et al., 2003; Orwa et al., 2009). This plant is drought resistant

like in southern part of Ethiopia and often grows in well-drained soils at altitudes of 900–1200 m

with a mean annual rainfall ranging from 500–1400 mm (Orwa et al., 2009), however, the cold

temperature is the limiting factor for the cultivation of this species.

7
2.2 Nutritional composition of Moringa plant

Moringa leaves have been advocated as a source of highly digestible proteins with considerable

amino acid profile that contains, the sulfur-containing amino acids, methionine and cysteine
(Booth and Wickens, 1988). It is also rich in the minerals calcium and iron and the vitamins A,

B, C and E (Booth and Wickens, 1988). The leaves are also rich in β-carotene and are an

exceptionally good source of fiber (Nambiar et al., 2003). M. stenopetala leaves are rich in

protein (28.2%) and contain reasonable amounts of essential amino acids, vitamins, and minerals

(Abuye et al., 2003; Melesse, 2011). M. oleifera leaves are nutritious and contain an average of

29% protein, 28 mg of iron, 1.9 g of calcium, and 0.8 g of vitamin C (Wangcharoen and

Gomolmanee, 2013). The leaf also has a wide range of beneficial polyphenolic compounds,

which include zeatin, quercetin, β-sitosterol, caffeolquinic acid, rutin, lutein, catechins,

isothiocynates and kaempferol (Nambiar et al., 2003). The seed also has a high oil (41.1%) and

protein content (42.6%) (Seifu, 2012). This shows that it can be used as a source of oil. In

addition, the seeds of this plant are used for water treatment (Bichi et al., 2012).

On the other hand, moringa leaf contains various antinutritional factors that may affect efficient

utilization, absorption and digestion of nutrients, and thereby decrease their bioavailability and

nutritional value (Lestienne et al., 2007). In addition, the bitterness and dark green color (Abuye

et al., 2003) is a limitation to the use of moringa leavf powder in food formulations. According

to the findings of Sengev et al. (2013), wheat bread with 5% of moringa leaves flour was

unacceptable though it has high nutritional values. Moreover, a wheat cookies developed by

mixing moringa leaves flour, had an acceptability to the maximum of 10% moringa flour mixture

(Nwakalor, 2014).

8
2.3 Bioactive compounds

In recent years, the possible toxicity and carcinogenicity of synthetic antioxidants have been
considered and make feasible the exploration of potential plants that can produce antioxidants

(Hou et al., 2003). Natural antioxidants protect against various diseases, which are induced by

free radicals. Antioxidants have various activities in addition to scavenging free radicals. These

include inactivating metal catalysts by chelation, reducing hydroperoxides into stable hydroxyl

derivatives and interacting synergistically with other reducing compounds (Frankel and Finley,

2008). The increased popularity of natural food additives may prompt food manufacturers to

replace synthetic antioxidants with ingredients containing natural anti-oxidative compounds.

These views make researchers to devote their efforts on natural antioxidants that are perceived as

posing no health risk to the consumers (Shi, 2006). Recent epidemiological studies indicate that

diets rich in fruits and vegetables and those of selected natural antioxidants, such as plant

polyphenols, vitamin C and flavonoids correlate with reduced incidence of chronic diseases like

cardiovascular and certain cancers (Siddhuraju and Becker, 2003). The importance of antioxidant

consumption is emphasized in the phenomena known as the French paradox. It is known that

people in certain districts of France have a diet with significantly high levels of saturated fat and

relatively high plasma cholesterol levels, but they have a low heart disease mortality rate (Renaud

and de Lorgeril, 1992). These phenomena are believed to be due to the fact that in these districts

of France, there is a high consumption of red wine and hence an associated high level of

phenolic/antioxidant intake (Meyer et al., 1997). The latter has been implicated to inhibition of

oxidation of low-density lipoproteins in the blood (Lila, 2004).

9
Antioxidants have known to be beneficial to the health of the consumers (Pandey and Rizvi,

2009). However, conventional processing of foods degrades and may also destroy the anti
oxidative properties of the foods. As a result, the utilization of synthetic antioxidants, such as

butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene (BHT) is increased (Shi, 2006).

However, these compounds claimed to have carcinogenic effects (Clevidence et al., 1997). This

drawback brings researchers to focus on the extraction and utilization of antioxidants from

natural sources. The sources of antioxidative compounds are fruits, vegetables, spices and others

by which plants can withstand a harsh environment. These compounds include phenolic acids,

flavonoids, isoflavones. tannins, lignans, coumarines, stilbenes, flavanones, and oligomeric

proanthrocyanidins (Shahidi, 1997). In addition, the known natural antioxidants that have been

proven to be beneficial to human health are tocopherols, vitamin C and carotenoids, polyphenols

and flavonoids (Siddhuraju and Becker, 2003).

Moringa has a wide range of beneficial bioactive compounds which include polyphenolic acids,

flavonoids, glucosinolates and others(Bennett et al., 2003; Habtemariam, 2015). Total phenolic

content of aqueous Moringa leaves extracts is 45.81 mg GAE/g (Sreelatha and Padma, 2009),

48.36 mg GAE/g (Das et al., 2012), and the total flavonoid content is about 27 CAE mg/g

(Sreelatha and Padma, 2009). These indicate that Moringa leaves extracts have a high content of

bioactive compounds. Since polyphenols are responsible for the antioxidant activity, moringa

leaves extract can be potential source of bioactive compounds. This makes the leaves extract to

possess various biological activities, including hypocholesterolemic, antidiabetic, hypotensive

agent (Nardos et al., 2011; Toma et al., 2012); antihypertensive activity (Dangi et al., 2002;

Geleta et al., 2016).

10
2.4 Extraction techniques of bioactive compounds
Organic solvents like ethanol, ethyl acetate, methanol, dichloromethane and others are used for

selective extraction of specific constituents from plant products; example isolation of bioactive

components from hops, spices, oil seeds, and other plant products (Shi, 2006). Extraction

solvents must meet the legal requirements put in place to ensure food quality and safety. These

requirements are a high degree of purity, chemical stability, inertness, low boiling point, and no

toxic effects. The criteria in these regulations are set by national and international bodies such as

the US Food and Drug Administration (FDA); European Economic Commission (EEC); Codex

committee; the Canadian Food Inspection Agency (CFIA); and FAO/WHO Codex Alimentarius

Commission (Feng et al., 2011). Most countries have regulations stating which extraction

solvents are generally regarded as safe (GRAS) (Shi, 2006). However, some chemicals that are

used as a solvent extraction medium can be toxic and cause other disorders when they are

inhaled. Because of these reasons, conventional solvent extraction methods are viewed with

suspicion concerning their role in the development of functional foods (Shi, 2006).

Ulrasonication extraction is generally considered as safe, non-toxic, and environmental friendly,

thus ultrasound has an advantage over conventional extraction techniques (Feng et al., 2011). A

potential new technology that improves the extraction of active compounds from plants at high

intensity ultrasound intensity has been reported (Li et al., 2004). High-intensity ultrasonication

can accelerate heat and mass transport in a variety of food processing operations and has been

successfully used to improve efficiency of extraction (Feng et al., 2011). Ultrasonication is the

application of high-intensity, high-frequency sound waves and their interaction with materials

(Luque-Garcıa and De Castro, 2003). The propagation and interaction of sound waves alter the

11
physical and chemical properties of materials, which are subjected to ultrasound (Mason et al.,

1996). Ultrasound causes the disruption of the plant cell wall, thereby facilitating the release of

extractable compounds and enhance mass transport of the solvent from the continuous phase into

plant cells (Vinatoru, 2001).

The pomegranate seed oil yield extracted by using ultrasonic-assisted extraction is significantly

higher than Soxhlet extracted and supercritical fluid extraction (Tian et al., 2013). The yield of

ultrasound extracted saponin from ginseng was increased by 15% at a yield of saponin by 30%

(Li et al., 1994). Extraction of pyrethrines from pyrethrum flowers and oil from woad seeds was

also enhanced (Romdhane and Gourdon, 2002); moreover, the acceleration of extraction kinetics

with the ultrasonicator was noted as well as an increase of the yield. Improved yields of

lipophilic compounds extracted from herbs such as Coriander and Fennel have been reported

(Vinatoru, 2001). According to Li et al. (2004), the soybean oil yield is increased when the

intensity of ultrasound increased. Therefore, it is concluded that ultrasonic assisted extraction has

an impact on the yield of bioactive compounds.

2.5 Encapsulation for food industry

Encapsulation may be defined as a process to entrap one substance within another substance,

thereby producing particles with diameters of a few nanometers to a few millimeters.

Microencapsulation considered as the packaging technology of solids, liquid or gaseous material

with thin polymeric coatings, forming small particles called microcapsules. They can release

their contents at controlled rates under specific conditions (Gharsallaoui et al., 2007). The

polymer acts as a protective film, isolating the core and avoiding the effect of its inadequate

exposure to environmental stresses (Desai and Jin Park, 2005). The substance that is
 12
encapsulated may be called the core material, the active agent, fill, internal phase, or payload

phase, whereas the substance that is encapsulating may be called the coating, membrane, shell,

carrier material, wall material, external phase, or matrix. The carrier material of encapsulates

used in food product should be of food grade and able to form a barrier for the active agent and

its surroundings, thus making it released at the ideal place through a specific stimulus (Zuidam

and Shimoni, 2010).

Encapsulation has been used in the food industry for coating food ingredients such as flavors,

antioxidants, colors, acidulants, probiotics, polyunsaturated fatty acids, enzymes, vitamins (Pegg

and Shahidi, 2004). This encapsulation application uses to prevent active ingredients from

environmental factors: temperature, oxygen, moisture, pH and light (Desai and Jin Park, 2005).

Therefore, encapsulation improves the shelf life, control the release of the active ingredient under

specific conditions, masking of undesirable taste and odor, providing dilution and uniform

dispersion of the active ingredient and easier handling and transportation (Gharsallaoui et al.,

2007).

Generally, capsules are classified according to their size: macrocapsules (>5,000 μm),

microcapsules (0.2 to 5,000 μm), and nanocapsules (<0.2 μm) (Zuidam and Shimoni, 2010). In

terms of their shape and construction, the capsules can be divided into two groups:

microcapsules and microspheres. Microcapsules are particles consisting of an inner core,

containing the active substance, which is covered with a polymer layer constituting the capsule

membrane. Instead, microspheres are matrix systems in which the core is uniformly dispersed

and/ or dissolved in a polymer network (Gharsallaoui et al., 2007). Microspheres may be

 13
homogeneous or heterogeneous depending on whether the core is in the molecular state

(dissolved) or in the form of particles (suspended), respectively; (Sagis, 2015).

Commercialized encapsulated products developed by Orbitz Company, found in Canada, sells a

drink containing a suspension of colored capsules containing different aromas and some

vitamins. Moreover, in USA the company ‘Salvona’ has developed encapsulation technologies

allowing sequential release of aromas and sensory active ingredients in functional foods

(Poncelet et al., 2011). However, in our country, there are no such products even though there is

a demand. There are processed products from moringa in powder form, oil, seed cake and tea

(Mishra et al., 2012). However, these products have a bitter taste, and loose the bioactive

compounds upon storage; consequently, it may loss the health effects. Therefore, encapsulation

can be an option for eliminating these problems.

2.6 Benefits of encapsulation in food processing

Encapsulation has numerous applications in areas such as the pharmaceutical, agricultural,

medical and food industries; they are widely used in the encapsulation of essential oils,

colorings, flavorings, sweeteners, microorganisms (Sagis, 2015). Encapsulation has many

possible benefits in the food industries such as easier handling of the active agent, immobility of

active agent in food processing systems, improved stability, masking the undesirable taste and

odor and controlling the release of the active compounds (Zuidam and Shimoni, 2010). These are

beneficial to minimize costs, complexity of the production process and/or supply chain,

undesirable consumer perception (visual or touch) of the encapsulate, and stability challenges of
the active compounds during processing and storage (Sagis, 2015). Encapsulated bioactive

14
compounds can be consumed directly or by mixing with other food materials in composite

functional food product matrix.

Moringa leaves were reported to possess anti-atherosclerotic and antioxidant properties (Chumark

et al., 2008; Verma et al., 2009). The major phytochemical constituents in the leaves are phenolic

compounds and flavonoids (Habtemariam, 2015) such as cryptochlorogenic acid, isoquercetin

and astragalin (Vongsak et al., 2012). These compounds are well known for their wide range of

activities, including antioxidation, antihypertension and antinflammatory characteristics (Geleta

et al., 2016; Toma et al., 2012; Vongsak et al., 2013a). Because of these abundant medicinal

benefits, nutraceutical products could be developed from this plant. According to Vongsak et al.

(2013b) report, the deterioration of antioxidant compounds and free radical scavenging activity

of moringa leaves extracts were deccelerated when stored under high temperature and humidity.

Even if it is recommended to store the leaves extract in dry and cold place by Vongsak et al.

(2013b), encapsulation may be the one solution to keep the qualities and health effect of the

moringa leaves extract.

2.7 Selection of encapsulating materials for food industry

Among the different encapsulating/ coating materials generally used the most common to include

carbohydrates and their derivatives such as alginate, pectin, amylose, carrageenan, cyclodextrin,

inulin, chitosan; proteins and their derivatives (whey and soybean proteins isolates, casein);

synthetic polymers like ethyl cellulose, lecithin and others (Quek et al., 2015). The selection of
wall materials has considerable effects on the properties of the microencapsulated product.

Generally, wall materials are required to possess some of the following characteristics: film

former, flexible, tasteless, non-hygroscopic, soluble in an aqueous medium or solvent, and also

15
able to exhibit a phase transition, like melting or gelling (Gibbs, 1999; Schrooyen et al., 2001).

Moreover, encapsulating materials that are used in the food industry should be easily digestible,

have no interaction with the core material, be impermeable to water, inexpensive, not bringing

sensorial changes and be suitable with food regulations and local customs (Gibbs, 1999; Gouin,

2004; Schrooyen et al., 2001).

Based on these criteria numerous coating materials have been employed to encapsulate active

ingredients. Most of them are natural or derivatives of plant or animal food materials, which have

been approved by FDA (Zuidam and Shimoni, 2010). More importantly, these materials are

relatively inexpensive and widely used in numerous applications due to their well-known

physical properties (Cilek et al., 2012; Zuidam and Shimoni, 2010).

Maltodexrins are the most commonly used wall materials for encapsulation; there are of different

dextrose equivalents, as obtained by partial hydrolysis of starch, using acid and enzymes (Gibbs,

1999). The advantages of maltodextrin for microencapsulation are the relative low cost, high

solubility, low viscosity at high solids concentration, neutral flavor and taste (Krishnan et al.,

2005). Moreover, it forms a colorless solution and provides good protection of the core from off

flavor development due to oxidation; as a result, these materials are used extensively in the food

industry (Gibbs, 1999; Krishnan et al., 2005). The limitation of maltodextrin as a coating

material is its low emulsifying capacity and marginal retention of volatiles that can be improved
by mixing with other wall materials having a hydrophobic function (Krishnan et al., 2005).

Pectins are complex polysaccharides that can be grouped into two categories based on their

content of a methoxylated carboxyl groups; i.e. degree of esterification (DE) (Van Buren, 1991).

These are high methoxyl pectins (DE > 50%) and low methoxyl pectins (DE < 50%). High

16
methoxyl pectins form gels through hydrogen bonding between free carboxyl groups and

hydrophobic interactions between methyl esters (Taylor, 2012) that requires acidic conditions

(lower pH). The gelling ability of pectins makes it an ideal agent to make a biodegradable

hydrogel beads, films and coatings for microencapsulation purposes (Burey et al., 2008; Liu et

al., 2007). Since the high methoxyl pectin stability and hydration rate is dependent on pH

change, pectins can be used mostly in a pH dependent delivery system (De Yao et al., 1996; Liu

et al., 2007). When the pH < 4, the solubility of this pectin is poor while it dissolves highly when

the pH >7.0 (Vaidya et al., 2009).

Instead, the solubility of low methoxyl pectin is relatively high at acidic pH (Mura et al., 2003).

Since pectin is not digested by human digestive enzymes that give advantages to have site

specific delivery for the bioactive compounds, whereas the pectin matrix can be considered as a

dietary fiber source (Wong et al., 2011). Thus, pectin is important as a coating material to

substitute others; pectin is a relatively low cost ingredient when selected for encapsulation by

substituting other coating materials like proteins and gum arabic which are more expensive

(Drusch, 2007).

2.8 Encapsulation technologies

There are different technologies applied for encapsulation of bioactive compounds. These are

spray drying, coacervation, liposomes, inclusion complexation, co-crystallization, nanoparticles,

freeze-drying, and emulsion technologies (Fang and Bhandari, 2010; Gharsallaoui et al., 2007).

Each of these methods has its own specific strengths and limitations in encapsulation efficiency,

storage stability, control release properties, processing cost and production, ease of use,

biodegradability, and biocompatibility (Quek et al., 2015). Spray drying is among the most

17
commonly used methods by drying a liquid feed through a hot gas stream of high velocity

(Papoutsis et al., 2018). The spray drying process comprises four stages such as feed

atomization; contact the feed with the air stream, evaporation of water, and separation of the

dried product from the drying air (Papoutsis et al., 2018). The liquid feed is pumped through an

atomizer device that produces fine droplets into the main drying chamber (Gharsallaoui et al.,

2007), followed by the contact with the circulating hot air. Thus, the coating material

encapsulates the active compounds as water is evaporated. The spray drying process is affected

by different parameters such as inlet air temperature, feed flow rate and aspirator rate, and also

nature of the core coating materials (Desai and Park, 2005). Spray dryer is extensively used to

produce powdery products.

 Figure 2.1: Microencapsulation process flow diagram.

There are spray-drying parameters that can affect the physicochemical properties of the product;

i.e. inlet air temperature, feed rate, residence time (Gharsallaoui et al., 2007). As described by

Paini et al. (2015), the moisture content was lower when the feed rate of the spray dryer was

18
lower. This is due to the longer the residence time of the solution in the chamber when the feed

rate is lower. The advantages of this process are relatively low in cost and ease of scale up

(Gharsallaoui et al., 2007). However, the suitable shell materials for this process is the limiting

factor, and this technique use generally low payload (<40%) and problems with heat-sensitive

materials (Gouin, 2004; Madene et al., 2006).

Freeze-drying, also called lyophilization, is one of the most useful processes for drying heat

sensitive compounds in aqueous solutions. It involves the sublimation/removal of water from the

dispersed solids. The food industry widely uses this technology to preserve plant or animal

products in dehydrated powder forms. In the case of microencapsulation operation, it can be used
to dehydrate and convert food emulsions into powders (Cilek et al., 2012). The technique is

relatively simple and can provide better particle properties compared to spray drying and

drum/tray drying, such as resistance to oxidation and intact shape of microcapsules (Madene et

al., 2006). However, it requires longer processing time for dehydration depending on the

materials and the loads (Desai and Park, 2005).

2.9 Concluding remarks

Moringa stenopetala, often referred to as the African Moringa tree, is a multipurpose tree, in that

it has nutritional values and medicinal properties, and can be used for oil and water clarification.

It is native to Ethiopia, Northern Kenya, and Eastern Somalia areas with arid and humid climates.

Moringa stenopetala is abundantly present in the southern part of Ethiopia, where it is originated

and indigenously used as food and disease treatment remedies. Therefore, the utilization of the

indigenous plant, M. stenopetala, for the production of bioactive compounds is very important

for industrial and other potential utilization. In Ethiopia, the Moringa tree is

19
mainly cultivated and now it has expanded into different areas, which in turn increases the

biosynthesis potential for bioactive compounds and reduces the burden on highly consumed

plants; i.e. providing an alternative bioactive source.

Bioactive compounds are mostly secondary metabolites of plants, which are produced to increase

their overall ability to survive and withstand the harsh environments and prevent their infection

from diseases. The leaves of moringa are nutritious and possess bioactive secondary compounds

with antioxidant properties that may exert protective effect chronic diseases such as diabetes,

antihypertensive and antihyperglycemic activities and for others. Research findings on the
medicinal use of M. stenopetala further support the traditional claim of this plant particularly,

with respect on its use on treatment of chronic diseases. M. stenopetala, is an underutilized plant

whose bioactive compounds can be valorized by transforming them into products to maximize its

beneficial effects. Most of the natural bioactive compounds used are antioxidants in a plant,

which can formulate antioxidant extracts. However, such natural antioxidants are very unstable.

Therefore, microencapsulation can be used to protect the active compounds during processing

and storage conditions to minimize the loss of their bioactivity and improve their bioavailability

by controlled release. Therefore, the development of products with inclusion of bioactive

compounds will create a good opportunity for manufacturing entrepreneurs and may additionally

have economic benefits for the local communities.

20
Chapter 3

General Materials and Methods

3.1 Sample collection, transportation and preparation

The samples of M. stenopetala leaves were collected from Arba Minch, located at 6°01'59" N

and 37° 32'59" E, at a distance 505 km from Addis Ababa, Ethiopia. The altitude of the

collection site is about 1285 meters above sea level with an average temperature range of 24-
34°C. The collected specimen was identified by the botanist, and deposited in the herbarium of

traditional and modern medicine research directorate, Ethiopian Public Health Institute with

voucher number Debebe W.001.2016. The collected samples were washed immediately and

rinsed using distilled water to remove any foreign matter. Then, the samples were dried, ground

and allowed to pass through a sieve (20 meshes). Finally, the samples were packed, sealed and

stored in the dark at ambient temperature until extraction was carried out.

3.2 Chemicals and reagents

All chemicals and reagents used were analytical grades. These chemicals were ethanol,

methanol, ferric chloride, aluminum chloride, potassium persulfate, sodium nitrite, ferrous

sulfate, acetic acid, sodium acetate trihydrate, sodium carbonate, ethyl acetate and hydrochloric

acid (Dae Jung, Gyeonggi, Korea). Rutin, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH),

Folin-Ciocalteu’s Phenol reagent, ferrozine, 2,2’-azino-bis(3-ethylbenzothiazoline-6-

sulfonicacid) diammonium salt (ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), sodium

hydroxide, Ethylenediaminetetraacetic acid (EDTA), catechin, and 6-hydroxy-2,5,7,8-

21
tetramethylchroman-2-carboxylic acid (Trolox), monobasic potassium phosphate, pancreatin and

pepsin (Sigma-Aldrich, Germany).

3.3 Moisture content determination

The moisture content of the fresh leaves sample and the dried samples were determined

according to the method described by AOAC (2000). About ten grams of the fresh sample were

taken randomly from the stock and dried at 105 °C for 6 h in the oven (OV-150C, Genlab,
England). The average values were taken from triplicate experiment and percent of the moisture

content was determined using the equation 3.1.

Where, MC is the
moisture content (%), Wf is the weight of fresh sample (g), Wd is the weight of dried sample (g)

3.4 Extraction Methods

3.4.1 Convectional (Maceration) extraction

Extraction of M. stenopetala leaves was done according the method described by Vongsak et al.

(2013) with modification. Briefly, 25 g of the powdered dried samples of M. stenopetala leaves

was weighed. Samples prepared using each drying methods were kept in a sealed glass flask,

covered with aluminum foil to avoid the contract to light, and macerated with 70% ethanol, 50%

ethanol and 100% aqueous systems with occasional shaking at 170 rpm using orbital shaker

(VWR DS-500, USA) for 72 h. The extractions for 70% and 50% ethanol were obtained

exhaustively by filtration of the extracts using every 24 h through changing new solvent. The

22
aqueous extracts were undertaken using distilled water for 6 hours. The extracts were

subsequently filtered using double filter cotton-made clothes, kept overnight in refrigerator and

dried using a lyophilizer (LABACON, Freezone 6, Kansas City, USA). All extractions were

carried out at ambient temperature (24–28 °C). The hydro-ethanolic extracts were concentrated

and dried using a rotary evaporator (R-200, BÜCH Rotavapor, Switzerland) and stored at 4 °C

until analysis.

3.4.2 Ultrasonic-assisted extraction

Extracts were obtained from the dried M. stenopetala leaves using ultrasonic-assisted extraction

(UAE) by modifying (UAE parameters) the method of Albu et al. (2004). The powdered sample

was mixed with a solvent (ethanol:water) in different ratios (%), sonication time (min) and a

solute/solvent ratio (g/mL). Then, the mixture was mixed from 10-90 min in an ultrasonic water

bath (ULTRAsonik104x, Ney Dental Inc. Yucaipa, CA, USA) at 47/48 kHz (100% power) at a

temperature range of 30 °C. Then, the extracts were filtered and evaporated using a rotary

evaporator. Finally, the concentrate was stored in a refrigerator at 4 °C until analysis. The

optimization of extraction was also done based on the bioactive yield and the antioxidant activity

obtained.

3.5 Quantitative analysis of bioactive compounds and antioxidant activities of the extracts

3.5.1 Determination of total phenolic contents

The total phenolic content (TPC) of the extracts was quantified spectrophotometrically using the

Folin-Ciocalteu total phenol determination procedure outlined by Singleton and Rossi (1965). A

0.5 mL of the extract, 0.5 mL of standard gallic acid and the vehicle used as a blank were

23
transferred into three different test tubes. Then, 2.5 mL of 10% aqueous (v/v) Folin-Ciocalteu

reagent was added to each of the test tubes and mixed well using a vortex mixer. The mixtures

were kept for 8 min at room temperature and 2 mL of 7.5% (w/v) sodium carbonate in water was

added and mixed. The samples, standard and blank were then kept in the dark at room

temperature for 2 h. The absorbance was measured at 765 nm using a UV-Vis spectrophotometer

(Shimadzu, UV-1800, Japan). The concentrations of total phenolic compounds in the extracts
were determined by comparing the absorbance of the extract samples with that of gallic acid as

standard solutions. TPC was expressed as mg gallic acid equivalent per g leaves powder (mg

GAE/g dm) using the equation shown below.

Where, TPC- total phenolic content (mg GAE/g dried sample), c- concentration of gallic acid

established from the calibration curve (mg/mL), v- volume of the extract (mL), m- dry weight of

the leaves powder (g), d- dilution factor

3.5.2 Determination of total flavonoid contents

The concentrations of total flavonoid content (TFC) was determined according to the procedure

outlined by Adom and Liu (2002). A 0.5 mL of the extract and 0.5 mL of the catechin/rutin

standards were transferred in separate test tubes and mixed with 0.15 mL of 5% (w/v) sodium

nitrite and 2.5 mL of distilled water. The sample, standard and reagent mixtures were vortexed

and left for 6 minutes at room temperature to facilitate the reaction. 0.3 mL of 10% (w/v)

aluminum chloride was then added to each sample, mixed and kept for more 6 min. This was

followed by the addition of 1 mL of 1.0 M sodium hydroxide subsequently 0.55 mL of distilled

24
water. The mixture was vortexed and kept for 15 min. Finally, the concentration was measured

using a UV-Vis spectrophotometer at 510 nm. The TFC was expressed as mg catechin equivalent

per g of leaves powder (mg CE/g dm) as expressed above on the equation 3.2.

3.5.3 DPPH radical scavenging activity assay

The free radical scavenging activity of the M. stenopetala leaves extract was measured using the

2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the method described by Brand-Williams et

al. (1995). A 6 ×10-5 M DPPH stock solution was prepared using 2.4 mg of DPPH in 100 mL

80% methanol and the absorbance was measured using a spectrophotometer and checked that the

reading is less than one at 515 nm to ensure that the concentration of the reagent is optimum for

determination of the scavenging activity. 0.1 mL of the samples and standards of different

concentrations using 80% methanol mixed with 3.9 mL of diluted DPPH solution. The mixtures

were then mixed and kept in the dark place at room temperature for 30 min. The absorbance of

the ascorbic standard and samples were measured at 515 nm using a UV-Vis spectrophotometer.

The percent of inhibition (scavenging) activity was calculated using the formula shown below.

The IC50 values were also calculated.

3.5.4 ABTS radical scavenging assay

The 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS+) radical

scavenging assays were done according the method described by Re et al. (1999). In brief, ABTS

was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS +) was produced by

reacting 7 mM concentration of ABTS solution with 2.45 mM potassium persulfate and

25
incubated for 12 to 16 h in the dark at room temperature. This solution was then diluted using

methanol to get an absorbance of 0.70 ± 0.02 at 734 nm using UV-Vis spectrophotometer. After

optimizing the ABTS reagent 50 µl of sample extracts and standards of different concentrations
dissolved in 80% methanol were added to 5 mL of ABTS+ solution and mixed thoroughly. The

blank solution prepared using 80% methanol instead of the sample solution and assayed under

the same conditions. The absorbance was then measured after 6 min at 734 nm using UV-Vis

spectrophotometer. Then a percent of radical scavenging activity of the extracts and the standards

were calculated using the equation shown on the equation 3.3.

3.5.5 Ferric reducing power assay

The ferric reducing antioxidant power (FRAP) assay was done according to Nguyen et al.

(2015). First, the FRAP solution was prepared by mixing 300 mM acetate buffer solution

(pH=3.6), 10 mM of 2, 4, 6-Tris (2-pyridyl)-s-triazine (TPTZ) solution in 40 mM hydrochloric

acid and 20 mM ferric chloride solution in the ratio of 10:1:1, respectively. Then, 0.15 mL of the

sample was mixed with 2.85 mL of fresh FRAP solution. The mixture was then kept for 30 min

in the dark at ambient temperature. The same procedure was used for the blank and the Trolox

standard at different concentration. Then, the absorbance was measured using a UV-Vis

spectrophotometer at 593 nm. The reducing power was calculated from the Trolox standard

curve and expressed as mg of Trolox equivalent per gram of the sample (mg TE/g dm).

3.5.6 Ferrous ion chelating activity assay

This chelating activity of the extract was done based on the procedure described by Chew et al.

(2009). Briefly, 1 mL of 0.1 mM ferrous sulfate was added to 1 mL of the extract followed by

26
adding 1 mL of 0.25 mM ferrozine solution. The mixture was shaken vigorously and allowed to

stand for 10 min at room temperature in the dark. The same procedure was done for EDTA
standard with different concentrations and for the blank. Then, the absorbance was measured

using UV-Vis spectrophotometer at 562 nm. The ferrous ion chelating activity of the sample was

calculated and described as mg EDTA equivalent per gram of the sample using the EDTA

standard curve (mg EE/ g dm).

3.6 Experimental design and statistical data analysis

The effects of different drying methods and extraction solvents on the bioactive compounds were

evaluated by one-way analysis of variance (ANOVA) using a full factorial experimental design.

Optimization of extraction using ultrasonic-assisted extraction method was done. The design was

central composite rotatable design (CCRD) with three variables. These are sonication time,

solute solvent ratios and percentage of hydro-alcohol as a solvent that was studied on five levels

to investigate their effects on the yield of bioactive compounds and antioxidant activity.

Data were entered into JMP software (Version 13.0, 2016, SAS institute Inc., Cary, NC, USA) for

analysis. They were recoded, then categorized and sorted to facilitate its analysis. The mean and

standard deviations (mean ± SD) of the experimental findings were calculated. The analysis of

variance (ANOVA) was performed to determine significant differences between the means. The

Tukey HSD test was used to identify differences among samples. The difference between groups

with respect to variable under investigation was considered to be significant at significance level

P < 0.05. The results were presented in tables and graphs.

27
Chapter 4
Effects of Drying Methods and Extraction Solvents on Total Phenolic and

Flavonoids Content, and Antioxidant Capacity of Moringa stenopetala Leaves

Abstract

Moringa stenopetala is a multipurpose plant having nutritional and medicinal value. It is

claimed to be a rich source of essential macro and micronutrients as well as secondary

metabolites, which are essential bioactive components, hence it possesses health-promoting

activities. The aim of this study was to evaluate the effects of different drying methods and

extraction solvents on the bioactive compounds in M. stenopetala leaves extract. Different drying

methods, i.e. freeze-, shed-, room- and oven-drying were used; and different extraction solvents

were employed to determine the efficiency of extraction. The total phenolic content (TPC), total

flavonoid contents (TFC) and antioxidant activities were determined. Higher extraction yield was

obtained from freeze-dried samples extracted by 50% ethanol (34.94%) followed by shed

(34.18%) and oven-drying at 70 ºC and 60 ºC, respectively. There was a significant difference (P

< 0.05) among different drying methods and the extracting ability of the solvent ratios. Among the

drying methods, freeze-dried samples gave the high values of TPC (57.2 mg GAE/g dm) and TFC

(130.30 mg RE/g dm). The high antioxidant activity was observed in freeze-dried 70% ethanol

extracts with the IC50 values of 32.43 µg/mL and 13.11µg/mL for DPPH and ABTS radical

scavenging assays. This was followed by room-dried 70% ethanol extract with the IC50 values of

35.09 µg/mL and 15.97µg/mL, respectively. Therefore, 70% ethanol was found to be the best

extracting solvent to render high yield of TPC and TFC, which are the potential

28
bioactive compounds with antioxidant effects. The total yield of TPC and TFC, with antioxidant

effect were influenced by the different drying methods, type of solvent and the ratios employed for

extraction for M. stenopetala leaves.

Keywords: Moringa stenopetala, drying methods, extraction solvents, bioactive compounds,

antioxidant activities

4.1 Introduction

Moringa stenopetala has multipurpose uses such as source food, medicine, oils from seeds, water

clarification and biofuel production(Farooq et al., 2012). Currently, it has been observed that M.

stenopetala leaves are in the market as powder form, which is commonly used as herbal tea,

nutritional supplement as well as medicinal products. Moringa oleifera is the most extensively

studied species, while M. stenopetala, which is the most abundant indigenous species in

Ethiopia, is getting more attention to scientifically validate its wider traditional use.

As the nutritional value of Moringa varies due to different factors (Jiru et al., 2006; Melesse,

2011), the yield and antioxidant activities of Moringa extracts varies based on the geographical

location where it grows, type of varieties, stage of maturity and collection season (Bennett et al.,

2003; Habtemariam, 2015; Sreelatha and Padma, 2009). According to the recent report of

Habtemariam and Varghese (2015), the IC50 values of M. stenopetala which is an indigenous

source and the most abundant species of Ethiopia is over five times more potent than moringa

species abundantly present in India in terms of the phenolic compound constituents. This

indicates that M. stenopetala is far superior as a source of antioxidants than M. oleifera. M.

stenopetala contains higher amount of the flavonoid glycoside, rutin than M. oleifera suggesting
 29
that it may have a potential use as an alternative commercial source of rutin (Habtemariam,

2015).

Methods of drying of the fresh leaves can also influence the bioactive compounds yield and their

antioxidant activity (Chan et al., 2009; Nguyen et al., 2015). On the other hand, methods of

extraction, solvent type and solid solvent ratios have shown to influnce the extraction yield,

concentration of bioactives and antioxidant activity (Azmir et al., 2013; Do et al., 2014). This

could be exemplified by the reports on the effect of processing on cauliflower (Anwar et al.,

2013). When cauliflower was dried at 40 °C using oven dryer, it rendered the high yield of

antioxidants (30%), whereas, air-dried (at about 25 °C) cauliflower extracted by 80% methanol

gave the lowest yield (22%) (Anwar et al., 2013). This indicated that methods of drying and

extraction solvents could have a significant effect on the yield of constituents that have

antioxidant activity. In addition, different storage and processing methods can affect the stability

of the bioactive compounds (Sultana et al., 2008; Vongsak et al., 2013b). As a result,

manufacturers tend to use synthetic antioxidants such as Butylated hydroxytoluene (BHT) and

Butylated hydroxyanisole (BHA) that may be quite expensive and impart carcinogenic effects

(Clevidence et al., 1997), since the vital bioactive constituents from plant sources are lost due to

the processing and storage techniques employed.

It is therefore necessary to adopt appropriate drying methods in order to overcome the limitations

of processing and maximize the utilization of natural bioactive compounds. Drying is an

important process to reduce the moisture content of fresh materials to have longer shelf or

storage life of the sample without deterioration, besides reducing the cost of transportation that
preserves further processing. Different drying methods have showed significant influences on

30
stability of bioactive compounds and their antioxidant capacity in addition to their effects on the

physical and sensory quality (Dev et al., 2011; Nguyen et al., 2015). There are no so far any

reports on drying methods and extraction techniques for maximizing yield of the bioactive

components of M. stenopetala leaves. This study is therefore undertaken to determine the effects

of different drying methods and extraction solvents of M. stenopetala leaves on bioactive

compounds and the antioxidant capacity.

4.2 Materials and methods

4.2.1 Chemicals and reagents

All chemicals and reagents used were of analytical grades (Described in section

3.2) 4.2.2 Sample preparation for drying process

Drying of samples were performed using different drying methods, including freeze-drying,

oven-drying shed and room drying.

Freeze-drying – A portion of M. stenopetala leaves were kept in the freeze dryer (Alph 1-2LD

plus, Germany) for 48 hours.

Oven-drying – Samples were kept in the oven (OV150, Genlab, England) at 50, 60, and 70 °C)

and dried for different time periods.

Room drying – A portion of the sample was dried at an average room temperature and relative
humidity were 25 °C and 62%, respectively. The sample was dried for 72 hours. Temperature

31
and relative humidity were monitored using an iButton temperature/humidity data logger

(DS1923, iButton, San Jose, CA, USA).

Shed-drying – Portions of samples were kept under the shed at an average temperature and

relative humidity of 30 °C and 52% using the same setup indicated above. The samples were

ground, allowed to pass through a sieve (20 meshes), and kept in a sealed plastic bag protected

from light until extraction. Chemical analysis was then undertaken for total phenolics, flavonoids

content and antioxidant capacities on the processed samples prepared using different drying

methods and techniques.

4.2.3 Extraction of M. stenopetala leaves

Extraction was carried out as described in section 3.4.1 in aqueous, and 50% and 70%

ethanol. 4.2.4 Moisture content determination

The moisture content was determined (described in section 3.3).

4.2.5 Total phenolic contents

The total phenolic content was determined spectrophotometrically as described in section

3.5.1. 4.2.6 Total flavonoid contents

The total flavonoid content was determined as described in section 3.5.2.

4.2.7 DPPH radical scavenging activity assay

The free radical scavenging activity was measured as described in section 3.5.3.

32
4.2.8 ABTS radical scavenging assay

The ABTS radical scavenging assays were done as described in section 3.5.4.

4.2.9 Experimental design and statistical data analysis

The full factorial experimental design was used. The data were analyzed using JMP software

(Version 13.0, 2016, SAS institute Inc., Cary, NC, USA). All experiments were done in triplicate,

and expressed as mean ± standard deviation. The significance tests were performed by

comparison of means using ANOVA and differences among means were identified by the Tukey

HSD test. Differences were considered at a significance level of P < 0.05.

4.3 Results and discussion

4.3.1 Effect of drying methods on residual moisture content dried sample

The effects of different drying methods on the moisture content of the dried sample are shown in

Table 4.1. The samples were dried using different drying methods over time ranging from 10–72

h. The moisture content of the fresh sample was, on average 74.62%. The final moisture content

of the dried samples was in the range from 5.67–8.17%. Here, it was found that there were

significant differences in the moisture content of the sample dried by different methods.
Samples dried in an oven at 70 °C, 60 °C and 50 °C took relatively shorter drying time. On the

other hand, samples dried at room temperature took the longest time (72 h) when it was

compared to other drying methods. The variations of these drying time requirements might be

attributed to the factors such as, relative humidity, temperature and airflow rate of the

surrounding environment.

33
In general, shorter drying time could be achieved with lower relative humidity, higher

temperature and higher airflow rate. These conditions could be the possible contributing factors

for the differences in the drying time and methods among the processed samples. Shed drying

has the advantages of shorter duration compared to room drying, which may be attributed to the

lower relative humidity environment.

Table 4.1. Effects of different drying methods on the moisture content of M. stenopetala leaves.

Drying methods Drying time (h) Residual MC in dried samples (%) Freeze drying 24 6.67 ±

0.29c Room drying 72 7.17 ± 0.29bc Shade drying 48 7.67 ± 0.29ab Oven drying at 50 °C 21 5.83 ±

0.29d Oven drying at 60 °C 16 5.83 ± 0.29d Oven drying at 70 °C 10 5.67 ± 0.29d CBDT 72 8.17

± 0.29a a-dAll values are expressed as means ± standard deviation (n=3). Means with different

superscript letters in a column indicates significant (P < 0.05) differences among the drying

methods. CBDT- community based drying technique, MC- moisture content.

4.3.2 Effect of drying methods and extraction solvents on the extraction yield

There are different conventional and emerging technologies for extraction that can increase the

extraction yield from a plant material. Extract yield is not only dependent on the extraction

method, but also depends on type of solvents and the mixture ratios when more than one solvent
are employed during extraction. The yield of M. stenopetala leaves extract ranged from 24.46–

34
34.94% (Table 4.2). Higher extract yield was obtained for freeze-dried sample when extracted

with 50% ethanol. Higher extract yield was obtained mostly from 70% ethanol compared with

50% ethanol and water as extracting solvent (Tables 4.2). This finding is in in agreement with the

report of Do et al. (2014). The extract yield could be improved when a combination of alcohol

and water was used since both water and alcohol soluble compounds could solubilize the plant

constituents (Do et al., 2014).

Table 4.2. Effect of drying methods and extraction solvents on the extract yield from M.

stenopetala leaves.

Extracting solvents
Drying methods
70% ethanol 50% ethanol Aqueous

Freeze drying 33.72 ± 0.06cd 34.94 ± 0.02a28.45 ± 0.07k Room drying 32.79 ± 0.07f29.55 ± 0.51
j
33.20 ± 0.21e Shed drying 34.18 ± 0.02b26.21 ± 0.20 m32.42 ± 0.09f Oven drying at 50 °C 31.18

± 0.15g30.28 ± 0.45hi 27.50 ± 0.07l Oven drying at 60 °C 34.02 ± 0.14bc 24.46 ± 0.12n27.80 ±

0.10l Oven drying at 70 °C 34.10 ± 0.16bc 33.41 ± 0.20de 30.03 ± 0.16i CBDT 33.43 ± 0.05 de

30.61 ± 0.44h30.64 ± 0.44h a-nAll values are expressed as means ± standard deviation (n = 3).

Means with different

superscript letters in columns and rows indicate significant (P < 0.05) differences for different

drying methods and extraction solvent ratios. CBDT- community based drying technique.

The extract yield from freeze-dried sample was significantly (P < 0.05) higher than shed and

oven-dried samples (Table 4.2). The lowest extract yield (24.46%) was found from the oven
 35
dried sample at a temperature of 60 °C, which was extracted by 50% ethanol. This is better by far

than the extracted yield of M. stenopetala leaves powder (17.3%) extracted by absolute methanol

(Habtemariam, 2015). According to the report of Vongsak et al. (2013c), higher extract yield was

obtained for dried M. oleifera leaves in aqueous decoction (59.24%) than 70% ethanol extract

(40.5%). The extract yield increased for L. aromatica (33.67%) when using 50% acetone

compared to other extraction solvents (Do et al., 2014) and for C. reticulate L. a higher yield

(18.46%) was noted when 80% ethanol was used for extraction (Safdar et al., 2016). The

findings indicate that the extract yield of the plants strongly depends on polarity and the type of

solvents used (Sultana et al., 2007).

4.3.3 Total phenolic contents

As shown on Table 4.3, the total phenolic contents (TPC) were considerably affected by different

drying methods and extraction solvents. Thus, it had an impact on the antioxidant activities. It is

very important to determine relatively the best drying method that could preserve the phenolic

constituents without their deterioration. In addition, an appropriate extraction solvent must be for

efficient extraction of the phenolic constituents. Such measurments might facilitate the analysis

and identify the potential drying method and extraction solvent for nutraceutical product

development. According to this study, the TPC of the freeze-dried M. stenopetala leaves sample

extracted by 70% ethanol was 57.2 mg GAE/g dm, which was significantly (P < 0.05) higher

than the other drying methods. This result is in agreement with the report of Vongsak et al.

(2013c) who reported that the 70% ethanol extract gave relatively high TPC (53.5 mg CAE/g

dm) from M. oleifera leaves. Aqueous extracts prepared by oven drying at a temperature of 60 °C
had shown the second better yield of TPC (36.67 mg GAE/g dw) (Table 4.3).

36
The TPC of the samples was also significantly affected by different drying methods of

Phyllanthus amarus (Nguyen et al., 2015). In addition, the TPC of the aqueous extract from

oven-dried samples at a temperature of 50 °C and 60 °C was significantly higher than 70% and

50% ethanol extracts (Table 4.3). This showed that the drying methods, solvent types, and their

composition might have considerable effects.

Table 4.3. Effects of different drying methods and extraction solvents on the total phenolic

contents of M. stenopetala extracts (mg GAE/g dw).

Extracting solvents
70% ethanol 50% ethanol Aqueous
Drying methods

Freeze drying 57.2 ± 0.81a31.6 ± 0.40f22.67 ± 0.61n Room drying 31.87 ± 0.23f28.53 ±
hi
0.61 24.67 ± 0.61m Shade drying 32.8 ± 0.40e30.13 ± 0.46 g24.67 ± 0.61m Oven drying

at 50°C 33.6 ± 0.80cd 30.13 ± 0.23g33.73 ± 0.23c Oven drying at 60°C 25.73 ±

0.23l27.87 ± 0.23ij 36.67 ± 0.23b

Oven drying at 70°C 26.8 ± 0.40k28.93 ± 0.46 h27.07 ± 0.23k CBDT 32.93 ± 0.23de 26.93 ±

0.23k27.47 ± 0.92jk a-nAll values are expressed as means ± standard deviation (n = 3). Means with

different superscript letters in columns and rows indicate the presence significant (P < 0.05)

differences for different drying methods and extraction solvent ratios. GAE- Gallic acid

equivalent, dm- dry mass basis, CBDT- community based drying technique.

The TPC of the freeze-dried sample extracted using 70% ethanol showed a significantly (P <

0.05) higher value (57.2 mg GAE/g dm) when it was compared to the aqueous extracts that were
 37
oven dried at 60 °C and 50 °C. The aqueous extracts of freeze-dried samples had the lowest TPC

(22.67 mg GAE/g dm) compared to the other drying methods and extraction solvents. According

to the report of Assefa et al. (2015), a high TPC (44.86 mg GAE/g dm) was obtained from M.

stenopetala leaves prepared using aqueous decoction. Even if this showed the TPC of the sample

is depend on drying methods, extraction techniques employed may also maximize the phenolic

content of the extract (Safdar et al., 2016). Therefore, drying methods and time, solvent type

used for extraction and their ratios could affect the total phenolic contents (Nguyen et al., 2015).

Thermally driven drying methods such as sun, oven and microwave heating may also have a

negative effect due to the reduction of the total phenolic contents in the leaves (Chan et al.,

2009). The difference in total phenolic content of the extracts prepared from different drying

methods and extraction techniques might be attributable due to oxidation and decomposition of

bioactive compounds (Nguyen et al., 2015).

4.3.4 Total flavonoid content

M. stenopetala leaves samples dried with different drying methods have shown significant (P <

0.05) differences on the total flavonoid content (TFC) (Table 4.4). The high TFC was found in the

freeze-dried sample extracted by 70% ethanol (130.53 mg RE/g dm). The TFC of the room dried

sample, which was extracted with 70% ethanol was found to be 68.0 mg RE/g dm. The

shed dried sample had 65.07 mg RE/g dm followed by the oven dried samples at 50, 60 and 70

°C, which had 57.2, 41.33 and 34.53 mg RE/g dm, respectively (Table 4.4). This result indicates

that the TFC of M. stenopetala leaves extracts highly depends on the methods of drying and

extraction solvents. Similarly, the TFC of the extracts from freeze-, room-, shed- and oven drying
at 50 °C were found to be significantly lower when they were extracted by 50% ethanol

38
or water. These findings show that the TFC varies with the drying method employed and the

solvents used for extraction. The results are in agreement with those of Vongsak et al. (2013); the

TFC of M. oleifera leaves extract using 70% ethanol was found to be higher when it was

compared to other solvents.

Table 4.4. Effects of different drying methods and extraction solvents on the total flavonoid

contents of M. stenopetala extracts (mg RE/g dm).

Extracting solvent
Drying methods
70% ethanol 50% ethanol Aqueous

Freeze drying 130.53 ± 1.67a43.73 ± 0.46f14.00 ± 1.60m Room drying 68.00 ± 2.80b36.67 ±

0.61 g13.47 ± 1.01m Shade drying 65.07 ± 3.00c41.6 ± 2.27 f16.8 ± 1.60l Oven drying at 50 °C

57.2 ± 0.60d33.87 ± 1.40h28.4 ± 1.44j Oven drying at 60 °C 41.33 ± 2.44f31.2 ± 0.80i54.4 ±

0.40e Oven drying at 70 °C 34.53 ± 2.20gh 32.4 ± 0.40 hi 27.47 ± 0.83jk CBDT 53.6 ± 0.40e33.6

± 0.40hi 25.73 ± 1.85k

a-m
All values are expressed as means ± standard deviation (n = 3). Means with different

superscript letters in columns and rows indicate significant (P < 0.05) differences for different

drying methods and extraction solvent ratios. RE- Rutin equivalent, dm-dry mass, CBDT

community based drying technique.

Among the drying methods considered under this study, the freeze-dried sample extracted with

70% ethanol yielded the highest values of TFC (130.53 mg RE/g dm). On the other hand, the

TFC of the freeze-dried sample was found to be the least when the sample was extracted with
 39
water (Table 4.4). The aqueous decoction of the leaves of M. stenopetala for 5 minutes yielded

higher TFC (20.36 mg QE/g) when it was compared with 10 and 15 minutes decoction (Assefa et

al., 2015). This indicates that even if the drying methods have significant impact on the TFC of

M. stenopetala leaves extract, the solvent type and ratios used for extraction may also have a

significant effect for that particular dried sample. Therefore, selection of appropriate extracting

solvent types and composition ratios are essential to maximize the yield of TFC of the plant

extract, thereby optimize the process for nutraceutical products development.

Although the oven-drying method was shown relatively the fastest drying method, the total

phenolic contents and total flavonoid contents in the dried sample were low, in particular at 70 °C.

This is might be attributed to the higher temperature that leads to the decomposition of the

bioactive compounds as stated by Nguyen et al. (2015). Higher temperature may have an

advantage to remove moisture within a short time that prevents decomposition of compounds due

to the hydrolytic enzymatic reactions.

On the other hand, higher temperature could decompose heat sensitive bioactive compounds.

These results are in agreement with the findings of the persimmon fruit dried using oven drying

(Karaman et al., 2014). In other study, the Phyllanthus amarus leaves were dried using

microwave heatings (Nguyen et al., 2015). In addition, the lower total flavonoid contents was

found in the extract of Salvia officinalis plant dried at far-infrared radiation at 65 °C (Hamrouni

Sellami et al., 2013). Therefore, it is important to optimize the drying temperature and to select

appropriate protocol to minimize the decomposition of the bioactive compounds.

 40
4.3.5 DPPH radical assay

The antioxidant activities were assayed to identify the constituents of plants having a preventive

role against the risk of oxidative stress-related diseases that could be arose due to environmental

and physiological factors. The DPPH radical scavenging activity of the 70% ethanol extract in

increasing order were freeze-dried > room-dried > shed-dried > oven-dried at 50 °C > locally

dried > oven-dried at 70 °C > oven-dried at 60 °C. Likewise, the antioxidant activity of the 50%

ethanol extract also varied depending on the oven drying conditions (Table 4.5). This is in

agreement with the report of Anwar et al. (2013) that revealed the maximum antioxidant activity

in cauliflower dried at 40 ºC using an oven-drier. However, the least antioxidant activity was

found when it was air-dried at ambient temperature.

The antioxidant activity of the samples varied under different drying conditions and types of

solvents used for extraction. The lowest IC50 value was found for the freeze-dried sample

extracted by 70% ethanol (32.43 µg/mL) followed by the room-dried sample (35.09 µg/mL). This

indicates that the freeze-dried sample extracted using 70% ethanol showed higher antioxidant

activity when it was compared to 50% ethanol (58.75 μg/mL) and aqueous extracts (90.26

μg/mL) (Table 4.13). It was earlier reported that the IC50 value for the radical scavenging activity

of M. stenopetala leaves powder was 59.5 μg/mL when extracted using absolute methanol for 2

weeks period (Habtemariam, 2015). Besides, as reported by Assefa et al. (2015), the IC50 value

was 41.5 μg/mL for the aqueous decoction of M. stenopetala leaves powder.
 41
Table 4.5. Effects of different drying methods and extracting solvents on DPPH radical

scavenging activities of M. stenopetala leaves extracts (IC50 values in µg/mL). Extracting

solvents

Drying methods 70% Ethanol 50% Ethanol Aqueous Freeze drying 32.43 + 0.26a58.75 +

1.25g90.26 + 1.04p Room drying 35.09 + 0.04b60.00 + 0.35h81.55 + 0.97n Shade drying 37.85 + 0

.13c59.62 + 0.18gh 84.95 + 0.33o Oven drying at 50 °C 36.70 + 0 .06c39.91 + 0.26d72.40 + 1.36m

Oven drying at 60 °C 65.04 + 0 .87j46.26 + 1.20f40.04 + 0.32d Oven drying at 70 °C 63.30 +

0.48i43.38 + 0.16e81.76 + 1.44n CBDT 42.19 + 0.13e66.93 + 0.82k70.97 + 0.94l a-pAll values are

expressed as means ± standard deviation (n = 3). Means with different superscript letters in

columns and rows indicate significant (P < 0.05) differences for different drying methods and

extraction solvent ratios.IC50- amount of antioxidants required to inhibit 50% of the radicals.

CBDT- community based drying technique

On the other hand, M. oleifera showed an IC50 value of 62.94 µg/mL when it was extracted with

70% ethanol which was the lowest by far compared with other extraction methods, i.e., 122

µg/mL when extracted with 50% methanol for 3 days and 78.15 µg/mL for aqueous decoction for

5 min (Chumark et al., 2008). These findings indicated that the extraction techniques and

temperature have a significant impact of the antioxidant effect of the extracts. Apparently, M.

stenopetala exhibits higher antioxidant activity when compared with M. oleifera. Habtemariam

and Varghese (2015) reported that the antioxidant activity of M. stenopetala is five times more
potent than that of Indian moringa leaf extracts. The best extracting solvent among other solvents

42
used for M. stenopetala was found to be 70% ethanol. The extracting solvents caused variation

on the IC50 values for different plants. In Kedrostis foetidissima leaves extract, the increased order

of antioxidant effect was reported for different extracting solvents; i.e. higher value was found for

methanol followed by chloroform, aqueous, acetone and petroleum ether, respectively (Pavithra

and Vadivukkarasi, 2015). In addition, 80% methanol gave high antioxidant activity for Kinnow

peel extract followed by 80% ethanol (Safdar et al., 2016). On the contrary, ethanol extracts have

been shown to have higher antioxidant activity compared to methanolic extracts

(Do et al., 2014; Vongsak et al., 2013c).

The variation in the radical scavenging activity could be attributed to different drying methods

and extraction solvents employed for plant materials. In the current study, the antioxidant activity

of the extract is significantly (P < 0.05) influenced by the drying methods. It was also observed

that freeze-drying and room drying methods are the suitable drying methods for better

anti-oxidant effect of M. stenopetala compared to other drying methods.

4.3.6 ABTS radical assay

The chemical constituents of plant materials are complex in nature. This may create difficulties to

evaluate the antioxidant effects of the plant extracts using a single method. Therefore, ABTS was

used as an alternate approach for DPPH to evaluate the radical scavenging activity. It is found

that the DPPH antioxidant assay values varied from those of ABTS. This is also illustrated by the

report of Floegel et al. (2011) who stated that a high antioxidant activity of strawberry was found

when the plant extract was analyzed using the DPPH assay compared to the ABTS radical assay.
This was found to be vice-versa for blueberry extract. Therefore, the ABTS +

43
antioxidant evaluation technique was employed besides the DPPH radical scavenging activity in

the current study.

Table 4.6. Effect of different drying methods and extracting solvent on ABTS radical scavenging

activities of M. stenopetala leaves extracts (IC50 values in µg/mL)

Extracting solvents

Drying methods 70% Ethanol 50% Ethanol Aqueous Freeze drying 13.11 + 0.05a28.57 +

0.13h40.28 + 0.29n Room drying 15.97 + 0.12b28.23 + 0.16h35.29 + 0.31l Shade drying 17.16 +

0.07c24.86 + 0.16g38.23 + 0.43m Oven drying at 50°C 17.00 + 0.11c17.80 + 0.09d33.32 + 0.42i

Oven drying at 60°C 28.32 + 0.24h21.15 + 0.10f20.88 + 0.12f Oven drying at 70°C 34.48 +

0.07k19.58 + 0.04e41.02 + 0.77o CBDT 17.20 + 0.05c35.22 + 0.02l33.90 + 0.45j a-oAll values are

expressed as means ± standard deviation (n = 3). Means with different superscript letters in

columns and rows indicate significant differences at P < 0.05 for different drying methods and

extraction solvent ratios.IC50- amount of antioxidants required to inhibit 50% of the radicals.

CBDT-community based drying technique.

In this study, the ABTS radical scavenging activity of the extracts was found to be significantly

(P < 0.05) different among the various drying methods and extracts (Table 4.6). The freeze-dried

samples extracted by 70% ethanol showed the lowest IC50 values (13.11 µg/mL), which is

followed by room-dried (15.97 µg/mL), shed (17.16 µg/mL), oven-dried at 50 °C samples (17.00

µg/mL) (Table 4.6). Concerning the solvent type, 50% ethanol extracts showed the higher
 44
antioxidant activity for the oven-dried sample at a temperature of 50 °C followed by that of 70

°C. This showed that 50% ethanol extract of oven-dried samples could have a better antioxidant

activity. When the aqueous extracts were considered, oven-dried at 60 °C had better antioxidant

activity followed by oven-dried at 50 °C. The aqueous extract had a lower antioxidant effect

compared to other drying methods except oven-dried sample at a temperature of 60 °C. The

freeze-dried aqueous extract is the one that showed lowest radical scavenging effect (40.28

µg/mL) (Table 4.6).

The IC50 value of ABTS radical scavenging assay was lower when it was compared with the IC50

values of DPPH radical scavenging activity. This might be due to the presence of various types of

compounds with antioxidant effects that can be scavenged by ABTS rather than DPPH. Floegel

et al. (2011) reported that ABTS assay is better than DPPH assay for pigmented and hydrophilic

extracts. As a result, the ABTS assay shows significantly higher antioxidant activities for fruits,

vegetables and beverages (Floegel et al., 2011).

4.4 Conclusions

M. stenopetala leaves possessing high yield of bioactive compounds and antioxidant activities

when the samples were dried by freeze dryer. This might be due to thermal and chemical

degradation of bioactive compounds, which is less at low temperature. The lower total phenolic

and flavonoid content in the extract from oven-dried M. stenopetala leaves might be attributed to

the destruction of bioactive compounds. As for the extracting solvent, 70% ethanol rendered

higher yield of TPC and TFC compared to the aqueous and 50% ethanol extracts. Such
treatments show that they might be useful to obtain bioactive compounds to substitute the

commercial antioxidants for nutraceutical product development.

45
Chapter 5

Improving the Yield of Bioactive Compounds and Antioxidant Activities of

Moringa stenopetala Leaves by Optimizing Ultrasonic-Assisted Extraction

Abstract

Ultrasonic-assisted extraction (UAE) parameters were optimized in this study using response

surface methodology (RSM) to improve the total phenolic content (TPC), total flavonoid content

(TFC), and antioxidant activity assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-

azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays of M.

stenopetala leaves. According to the results, the optimal UAE parameters were extraction time

(26 min), ethanol concentration (68%), and solvent-to-sample ratio (42 mL/g). Based on these

optimal parameters, the predicted TPC and TFC values were 45.7 mg GAE/g dm and 41.36 mg

CE/g dm, respectively. The antioxidant activities of the extracts were assessed using DPPH and

ABTS assays and the IC50 values were 29.23 µg/mL and 13.37 µg/mL, respectively, whereas the

experimental values of the UAE extracts using optimal parameters were 45.75 mg GAE/g dm

(TPC), 39.2 mg CE/g dm (TFC), 30.62 µg/mL (DPPH), and 12.87 µg/mL (ABTS). These show

that the experimental values were very close to the predicted values. Therefore, the optimized

UAE is important for the extraction of M. stenopetala leaves with the advantages of shorter time

and lower cost.

Keywords: Optimization, ultrasonic-assisted extraction, bioactive compounds, antioxidant

activity, Moringa stenopetala leaves

46
5.1 Introduction

Bioactive compounds from M. stenopetala leaves can be used for the production of natural

products that can be used as food additives and could substitute the synthetic antioxidants that

may have carcinogenic effects (Atrooz, 2009; Clevidence et al., 1997). Conventional and novel

technologies exist for the extraction of bioactive compounds from plants. Conventional

extraction techniques includes maceration, Soxhlet extraction, decoction, and percolation. The

limitations of the conventional extraction methods are high energy consumption (more than 70%

of the total process energy), time consuming, and a higher consumption of harmful chemicals

(Chemat and Khan, 2011). Therefore, the food industry is exploring new technologies that use

green techniques to extract bioactive compounds, such as ultrasonic-assisted extraction (UAE),

microwave-assisted extraction, supercritical fluid extraction, subcritical water extraction, and

pulsed electric field. Novel technologies have advantages over conventional extraction techniques

in terms of shorter extraction time, higher yield with better activity, and lower solvent

consumption (Chemat et al., 2017; Do et al., 2014).

The advantages of ultrasonic extraction are that it is safe, non-toxic, accelerates mass transport,

and is a green technology (Chemat et al., 2017; Feng et al., 2011). It involves the application of

high intensity and sound wave frequency radiation, which interacts with other materials and

changes the physical and chemical properties of the sample and leads to cavitation effects
(Chemat and Khan, 2011; Luque-Garcıa and De Castro, 2003); further, explosion of cavitation

bubbles in the solvent and on the surfaces of the sample occurs (Chemat et al., 2017). These lead

to the accelerated release of the constituents from a plant matrix due to the disruption of the plant

cell wall (Chemat and Khan, 2011; Vinatoru, 2001). UAE gave significantly higher yield of

47
pomegranate seed oil than Soxhlet (20.50%) and supercritical fluid extraction (15.72%) (Tian et

al., 2013). The yield of the extract and saponin content from ginseng increased by 15% and 30%,

respectively (Li et al., 1994). Higher yields of pyrethrines from pyrethrum flowers (Romdhane

and Gourdon, 2002), lipophilic compounds from coriander and fennel (Vinatoru, 2001), soybean

oil (Li et al., 2004), flavonoid (Albu et al., 2004), phenolic compounds from peaches and

pumpkin (Altemimi et al., 2016), and antioxidant compounds from marjoram (Hossain et al.,

2012) were obtained using UAE. Therefore, it is important to optimize UAE parameters to

improve the yield of bioactive compounds. No studies have reported the UAE of M. stenopetala

leaves. Therefore, the aim of this study was to optimize the UAE parameters for improving the

yield of total phenolics and flavonoids and the antioxidant activity of M. stenopetala leaves

extracts using the response surface method (RSM) and by validating the predicted values.

5.2 Materials and methods

5.2.1 Sample collection

The M. stenopetala leaves sample was collected as described in section 3.1

5.2.2 Conventional extraction

Conventional extraction was performed as described in section 3.4.1.

5.2.3 Ultrasonic-assisted Extraction

The M. stenopetala leaves were extracted as described in section 3.4.2. The ultrasonic-assisted

extraction (UAE) parameters were as follows: extraction time (10-90 min), ethanol concentration

in water ranging from 50-90% (v/v), solvent-to-sample ratio ranging from 10:1 to 50:1 (mL/g).

48
5.2.4 Total phenolic contents

The total phenolic content (TPC) was determined as described in section 3.5.1.

5.2.5 Total flavonoid contents

The total flavonoid content (TFC) was determined as described in section 3.5.2.

5.2.6 DPPH radical scavenging activity assay

It was measured as described in section 3.5.3.

5.2.7 ABTS radical scavenging assay

The ABTS +radical scavenging activity was assayed as described in section 3.5.4.

5.2.8 Experimental design and statistical data analysis

For the optimization of UAE, the experimental results of the response surface design were

analyzed using the Design Expert software (State-Ease Inc., Version 7, Minneapolis, MN, USA,).

Three independent variables such as extraction time (A, min), ethanol concentration (B, %), and
solvent-to-sample ratio (C, mL/g) was used to analyze their effects on the dependent variables

such as the TPC, TFC, DPPH, and ABTS using the central composite rotatable design (CCRD).

The complete design consists of 20 experimental tests with 14 factorial points and 6 replications

of the central points (Table 5.1). Subsequently, the TPC, TFC, and antioxidant activity using

DPPH and ABTS assays were measured for the extracts. They were then optimized

simultaneously by RSM. The actual values used in the response surface analysis and the

corresponding parameter values are shown in Table 5.1. A quadratic polynomial equation

49
(Eq. 1) was selected to model the treatment effects and treatment interactions that were used to

predict the optimal extraction parameters:

Y = βo + β1A + β2B + β3C + β12AB + β13AC + β23BC + β11A2+ β22B2 + β33C2 (5.1)

Where, Y is the response variable; A, B, and C are the independent variables for the extraction

time, ethanol concentration, and solvent-to-sample ratio, respectively; β0 is a constant; β1, β2, β3

are linear coefficients; β12, β13, β23 are interaction coefficients, and β11, β22, β33 are quadratic

coefficients for extraction time, ethanol concentration, and solvent-to- sample ratio, respectively.

For the validation of the UAE, extraction was performed using the selected optimum UAE

conditions and then the TPC, TFC, and antioxidant capacity using the DPPH and ABTS radical

scavenging capacity of the UAE extracts were compared with those from the conventional

extraction. To compare the results, the means were compared using one way analysis of variance

JMP software as described in section 3.6.

5.3 Results and discussion

5.3.1 Fitting response surface models

The response surface model was constructed using UAE parameters to predict the TPC, TFC,

and DPPH and ABTS radical scavenging activity of M. stenopetala leaves extract. Table 5.2

shows the model’s summary statistics. The determination coefficient (R2) was in the range of

97.23–99.51%, which was closer to the adjusted R2(ranged from 94.73% to 99.06%). In addition,

the predicted R2ranged from 81.01% to 96.52%, which shows reasonable good agreement with

the adjusted R2 (Table 5.2).

50
Table 5.1. Experimental design with the observed responses of TPC, TFC, DPPH, and ABTS

radical scavenging capacity of the extracts.

A B C Responses Y
Ethanol Solvent-to (mL/g) GAE/g dm) CE/g dm) (µg/mL) (µg/mL)
Concentra sample DPPH ABTS
Run TPC (mg TFC (mg
Time (min)tion (%) ratio IC50 IC50

1 50 50 30 28.33 19.55 34.67 14.49 2 26 58 18 39.06 21.63 32.23 13.76 3 90 70 30 12 10.45

46.85 20.88 4 26 58 42 42.02 36.73 30.88 13.24 5 50 70 30 33.29 29.41 31.88 14.94 6 26 82 42
46.21 37.47 29.23 13.88 7 50 70 30 29.12 28.78 32.13 14.45 8 10 70 30 38.11 30.96 30.93 14.45
9 26 82 18 36.17 31.57 29.93 13.62 10 74 82 18 19.44 15.55 42.08 20.42 11 50 70 50 45.5 42.49
31.98 12.99 12 74 82 42 20.55 13.67 41.63 17.57 13 50 70 30 32.69 29.31 32.1 14.48 14 50 90
30 20.1 16.06 39.3 17.31 15 74 58 42 28.14 24.27 35.63 14.98 16 50 70 30 31.19 28.99 31.8
14.26 17 50 70 30 32.11 29.41 31.5 14.48 18 74 58 18 23.21 22.61 39.1 15.1 19 50 70 10 41.4
31.98 34.52 15.71 20 50 70 30 32.72 29.1 31.65 14.5 TPC- Total phenolic content (mg GAE/g
dm); TFC- Total flavonoid content (mg CE/g dm);

GAE- gallic acid equivalent; CE- catechin equivalent; dm- dry mass of the sample; DPPH and

ABTS- IC50 values of DPPH and ABTS radical scavenging activity (µg/mL)

51
This showed that the quadratic model was adequately fitted to the TPC, TFC, DPPH, and ABTS

data. The relative importance of the UAE parameters with respect to the TPC, TFC, and

antioxidant activity assessed was investigated to determine more accurately the optimum

combinations of the independent parameters using ANOVA. The ANOVA results for the

response surface quadratic model constructed using the UAE parameters for TPC, TFC, and

DPPH and ABTS scavenging activity are given in Table 5.2. The model F values in the range of

38.95–224.02 imply that the model is highly significant (P < 0.0001) for the TPC, TFC, and

DPPH and ABTS scavenging activity.

In this study, all parameters are significant except for the interaction effects of AC and BC (Table

5.2). Since the fitness of the model is evaluated by the lack of a fit test, the lack of fits was not

significant (P > 0.05) relative to the pure error. As cited by Liu et al. (2015), the quadratic

polynomial model is well fitted to the experimental values if the regression values show

significance and the lack of fit test is not significant. Therefore, the values indicate the feasibility

of the model to predict the variations accurately.

5.3.2 Influence of UAE parameters on total phenolic content

The TPC of the extract as a function of extraction time, ethanol concentration, and solvent-to

sample ratio was graphically represented in three-dimensional surface plots as shown in Fig. 5.1

(a–c) and Table 5.2. According to the results, the R 2of TPC was 0.9733. This is higher than that

of the TPC models reported by Hossain et al. (2012) for Origanum majorana L. leaves

(R2=0.917). The extraction time and ethanol concentration had a greater effect on the yield of

phenolic compounds than the solvent-to-sample ratio (Fig. 5.1a). As the UAE parameters were

changed, the TPC also changed.

 52
Table 5.2. Regression coefficients for the fitted quadratic polynomial model and analysis of

variance for the experimental results of TPC, TFC, IC 50 values of DPPH, and ABTS radicals.

TPC TFC DPPH (µg/mL) ABTS (µg/mL)

Factors (mgGAE/g dm) (mg CE/g dm)
Intercept 32 29.15 31.78 14.49 Linear
A- Extraction time -8.00a-6.44a4.61a1.98a B- Ethanol conc. -2.23c-0.79d0.91d0.79a C-
Solvent-to-solute
ratio 1.49d3.26a-0.73ns-0.9a Interaction
AB -2.30d-3.12a1.55d0.57b AC -2.00ns-2.70a-0.085ns-0.86a BC 1.86ns-2.86a0.43ns 0.26ns
Quadratic
A2-1.90c-3.03a2.28a1.04a B2-2.42c-4.04a1.69b0.45b C24.40a2.83a0.37ns-0.1ns R20.9733 0.9951
0.9723 0.9915
Adj R20.9492 0.9906 0.9473 0.9838 Pred R20.8322 0.9652 0.8101 0.9345 P-value <0.0001
<0.0001 <0.0001 <0.0001 F-value 40.44 224.02 38.95 129.56
C.V. % 6.60 3.07 3.18 1.8 Lack of fitness 1.25 4.33 1.35 2.82 TPC- Total phenolic content (mg
GAE/g dm); TFC- Total flavonoid content (mg CE/g dm); GAE- gallic

acid equivalent; CE- catechin equivalent; dm- dry mass of the sample; DPPH and ABTS- IC50 values of

DPPH and ABTS radical scavenging activity (µg/mL); Conc.-concentration. a Significant at P < 0.0001; b

Significant at P < 0.001; c Significant at P < 0.01; d Significant at P < 0.05 and ns Non significant.

53
Extraction time and ethanol concentration were the most significant factors affecting the TPC of

M. stenopetala leaves extract, whereas the solvent-to-sample ratio was also significant (P < 0.05)

(Table 5.2). While sonication time had a significant effect on the TPC of a yallow plant extract,

the solvent-to-sample ratio had no significant effect (Bashi et al., 2012). It was also observed

that, for a given time, the yield of the phenolic content gradually increased with increasing

ethanol concentration. This agrees with the findings of Ghafoor and Choi (2009), who stated that
the yield of phenolic compounds in grape peel extract increased significantly with an increase in

ethanol concentration (P < 0.001).

Therefore, increasing the extraction time leads to an increase of solvent diffusion due to the

increase of ultrasonic waves that break the cellular structure of the leaves, hence improving the

efficiency of mass transfer of phenolic compounds (Horžić et al., 2012). Subsequently, the yield

of phenolic compounds declined as extraction time was further increased (Fig. 5.1a and 5.1b). In

this study, among the UAE parameters, the least TPC yield was observed at 90 min, 70%, and 30

mL/g whereas the optimum was at a short extraction time (26 min) (Table 5.1). Although

increasing the extraction time generally increased the yield of phenolics in the extract, a longer

extraction time may result in the destruction of phenolic compounds due to the longer effects of

the ultrasonic wave (Chemat et al., 2017). The regression equations using coded levels of the

independent variables and response variable were as follows:

Y (TPC) = 32 - 8A - 2.23B +1.49C - 2.3AB-1.9A2- 2.42B2+ 4.4C2(5.2)

54
ab
 c

Figure 5.1: Response surface for the combined effects of extraction time and ethanol
concentration (a); ethanol concentration and solvent-to-solute ratio (b); extraction time and

solvent-to-solute ratio (c) on the total phenolic content (mg GAE/g dm) of the extract from M.

stenopetala extracts

55
5.3.3 Influence of UAE parameters on total flavonoid content

The influence of UAE parameters on TFC is shown in Fig. 5.2 (a–c) and Table 5.2. The time of

extraction, ethanol concentration, and solvent-to-sample ratio affected the TFC significantly. It

was observed that the TFC increased with increasing extraction time, ethanol concentration, and

solvent-to-sample ratio. The increase of the TFC is also seen in green tea extract when the

ultrasonic extraction time is increased (Das and Eun, 2018). This is due to the increase in time,

which enhances the destruction of the sample’s cellular structure. The extraction time showed a

greater effect on the TFC than ethanol concentration (Liu et al., 2015). However, based on Fig.

5.2 (a and b), the yield of flavonoid compounds decreased beyond a certain extraction time.

The excessive exposure to ultrasonic extraction may lead to the decomposition of the flavonoid

compounds. The TFC increased as the ethanol concentration increased. This indicates that the

flavonoids were highly dissolved as the ethanol concentration increased, implying that the

increase in polarity improved the TFC. This agrees with Liu et al. (2015), who stated that the

yield of flavonoids increased as the concentration of ethanol was increased from 40% to 60%. As

indicated by Mustapa et al. (2015), the extraction efficiency of bioactive compounds is affected

by solvent polarity and concentration, solvent-to-sample ratio, and time of extraction.

Regarding the solvent-to-sample ratio, the TFC increased as the solvent-to-sample ratio
increased. At a lower solvent-to-solute ratio, the equilibrium can be set before the maximum

extraction of bioactive compounds has taken place (Feng et al., 2011). The regression equation

using the coded levels of the independent variables and response variable was as follows:

Y (TFC) = 29.15 - 6.44A - 0.79B + 3.26C - 3.12AB- 2.7AC - 2.86BC - 3.03A2 (5.3)
- 4.04B2+ 2.83C2

56
ab

Figure 5.2: Response surface for the combined effects of extraction time and ethanol

concentration (a); ethanol concentration and solvent-to-solute ratio (b); extraction time and

solvent-to-solute ratio (c) on the total flavonoid content (mg CE/g dm) of the extract from M.
stenopetala extracts

57
5.3.4 Influence of UAE parameters on DPPH and ABTS radical scavenging activity

The antioxidant activity assessed using the DPPH and ABTS assays was significantly affected by

the linear, interaction and quadratic (P < 0.001) except for the interactions of the BC parameters

and the quadratic of C (P > 0.05). The R2of DPPH and ABTS radical scavenging activity was

0.9723 and 0.9915, respectively. This agrees with the results of Li et al. (2016) for O. fragrans

(R2= 0.9829) but the values were higher than those for pumpkin (R2=0.713) and peach (R2=0.70)

(Altemimi et al., 2016). The effect of the UAE time (min), ethanol concentration (%), and

solvent-to-sample ratio (mL/g) on the DPPH and ABTS radical scavenging activity of M.

stenopetala leaves extract is shown in Table 5.1. The effects of independent variables on the

responses of DPPH and ABTS are also shown in three-dimensional surface plots: Fig. 5.3 (a–c)

for DPPH and Fig. 5.4 (a–c) for ABTS. The radical scavenging activity increased with increasing

extraction time, ethanol concentration, and solvent-to-sample ratio. This study indicates that

relatively shorter extraction times could not increase the antioxidant activities because the bound

bioactive compounds such as bound phenolic compounds that exist need longer extraction times.

This agrees with Das and Eun (2018) who found that the antioxidant activity is increased in

green tea extract when ultrasonication time increased from five to twenty minutes.

However, the antioxidant activity decreased as the extraction time increased (Fig. 5.3a and Fig.

5.4a). The decrease in the antioxidant activity of the extract through prolonged extraction time

might be due to the destruction of active compounds by ultrasonic wave and excess heat
exposure. This is because the exposure of the active compound to high temperatures for a long

time and the exposure to air affect the antioxidant activity of the extracts due to the

decomposition of the active compounds (Azmir et al., 2013).

58
ab

Figure 5.3: Response surface for the combined effects of extraction time and ethanol

concentration (a); ethanol concentration and solvent-to-solute ratio (b); extraction time and

solvent-to-solute ratio (c) on IC50 values of DPPH (µg/mL) from M. stenopetala extracts.
59