FICUS DELTOIDEA EXTRACT
NOR RASHID AH BINTI AHMED
A thesis submitted in fulfilment o f the
requirements for the award o f the degree o f
Master o f Engineering (Chemical)
Faculty o f Chemical and Energy Engineering
Universiti Teknologi Malaysia
D edicated to my beloved husba^id, ^M ohd ^Nasrol ^Shar^nan T o my beloved umi and baba
ACKNOW LEDGEMENT
Alhamdullllah, all praise to Allah the creator o f this world. I am grateful to
Him that I have completed my research and thesis. Firstly, I would like to express my
most sincere gratitude to my husband, mom and dad who have always been with me
throughout the process o f completing this thesis. Thank you for always being my
supporter, adviser, motivator and inspiration to complete this project.
Secondly, I wish to express my appreciation to my supervisor, Dr Harisun
Yaakob, for the guidance, encouragement and advices in completing this research.
Her patience and assistance have helped and motivated me to accomplish my study
and thesis. Besides, I would like to bid appreciation to my co-supervisor, Assoc.
Prof. Dr Azila bt Abd. Aziz, who has been very supportive and encouraging me in
helping me understand this research.
Thirdly, a lot o f thank to my friends Mrs. Noor Hafiza and Ms N ik Nurul
Najihah for their moral support, suggestions, ideas and time for discussions. I also
would like to thank all the research officers o f Institute of Bioproduct Development
(IBD) especially M r Ismail and Mrs Muna, for their guidance and assistances. In
addition, a great appreciation to M r M ohd Khusyairy, the staff and technician of
Separation Laboratory o f Faculty o f Chemical and Energy Engineering, Universiti
Teknologi Malaysia for his contribution to complete this research. I am also indebted
to Ministry o f Agriculture and Agro-based Industry, Malaysia under NKEA Herbal
Research Grant Scheme for funding my master study. Thank you.
ABSTRACT
Vitexin, a marker compound present in Ficus deltoidea (F. deltoidea) exhibits
various phytochemical properties valuable for human health. The previous study has
reported lower process yield from the production of F. deltoidea. Thus, this study
aimed to obtain the highest yield whilst maintaining the vitexin quality by optimising
the processing parameters for water extraction and spray drying o f F. deltoidea using
the Box Behnken Design.
In the extraction process, the effect of extraction
temperature, extraction duration and solvent to raw material ratio on total solid
content and vitexin were investigated. Whereas, for the spray drying process using
spray dryer with drying capacity o f 1000 ml/h, the effect o f the inlet air temperature,
feed flow rate, air pressure and feed temperature were determined based on process
yield, vitexin and moisture content. In addition, the thermal effect o f spray drying
process on four bioactive compounds o f F. deltoidea; vitexin, total saponins, total
protein, and total polysaccharides were identified. The experimental data obtained
from the extraction and spray drying processes were analysed using the analysis of
variance. The optimum conditions o f the extraction process was achieved at 87 oC of
extraction temperature, 2.71 hours o f extraction duration and 19.12:1 g/g o f solvent to
raw material ratio with 8.64 mg/g o f solid content and 0.62 % vitexin. The yield of
the extract at this condition was 17.39 %. Whereas, the optimum condition for spray
drying process was achieved at 191 oC of inlet air temperature, 2 ml/min o f feed flow
rate, 28 psi o f air pressure and 25 oC o f feed temperature with 10.01 % o f process
yield, 0.42 % o f vitexin and 4.33 % o f moisture content. The spray drying heat does
not affect the total saponins and total polysaccharides. However, it led to more than
30 % and 50 % reduction o f vitexin and total protein. Therefore, response surface
methodology was successful in the optimization o f the processing parameters for the
extraction and spray drying processes to produce the highest yield and maintain the
quality o f F. deltoidea extract.
ABSTRAK
Vitexin, sebatlan penanda dalam Ficus deltoidea (F. deltoidea) mempamerkan
pelbagal slfat fitoklmla yang balk untuk keslhatan manusla.
Kajlan sebelum inl
melaporkan hasll proses yang rendah darlpada pengeluaran F. deltoidea. Oleh ltu,
kajlan ini bertujuan mendapatkan hasll proses yang tlnggl dan mengekalkan kualltl
vitexin dengan mengoptlmumkan parameter pemprosesan pengekstrakan air dan
pengerlngan semburan untuk F. deltoidea menggunakan Box Behnken Design. Dalam
proses pengekstrakan, kesan suhu pengekstrakan, tempoh pengekstrakan dan nlsbah
pelarut kepada bahan mentah terhadap jum lah bahan kerlng dan vitexin telah dlslasat.
Manakala, untuk proses pengerlngan semburan menggunakan pengerlng semburan
dengan kapasltl pengerlngan 1000 ml/h; kesan suhu udara masuk, kadar allran
masukan, tekanan udara dan suhu masukan dltentukan berdasarkan hasll proses,
vitexin dan kandungan kelembapan. Tambahan lagl, kesan pemanasan pengerlngan
semburan ke atas kualltl empat sebatlan bloaktlf dalam F. deltoidea; vitexin, jum lah
saponin, jum lah protein, dan jum lah pollsakarlda telah dlkenal pastl.
Data
eksperlmen yang dlperoleh darlpada pengekstrakan dan pengerlngan semburan F.
deltoidea dlanallsls dengan menggunakan anallsls varlans. Keadaan optimum untuk
proses pengekstrakan telah dlcapal pada 87 oC suhu pengekstrakan, 2.71 jam tempoh
pengekstrakan dan 19.12:1 g/g nlsbah pelarut kepada bahan mentah dengan 8.64 mg/g
jum lah ekstrak dan 0.62 % vitexin. Hasll ekstrak pada keadaan ini adalah 17.39 %.
Manakala, keadaan optimum untuk proses pengerlngan semburan dlcapal pada 191 oC
suhu udara masuk, 2 ml/mln kadar allran masuk, 28 psl tekanan udara dan 25 oC suhu
masukan dengan 10.01 % hasll proses, 0.42 % vitexin dan 4.33 % kandungan
kelembapan. Haba pengerlngan semburan tldak menjejaskan jum lah saponin dan
jum lah pollsakarlda. Walau bagalmanapun, la membawa leblh darlpada 30% dan
50% pengurangan pada vitexin dan jum lah protein. Oleh ltu, kaedah permukaan gerak
balas berjaya mengoptlmumkan parameter pemprosesan pengekstrakan dan
pengerlngan semburan untuk menghasllkan hasll proses yang tertlnggl dan
mengekalkan kualltl ekstrak F. deltoidea.
TABLE OF CONTENTS
CHAPTER
TITLE
DECLARATION
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
LIST OF SYMBOLS
LIST OF APPENDICES
PAGE
v
vi
vii
xl
xlv
xvll
xvlll
xlx
INTRODUCTION
1.1
Background o f Study
1.2 Problem Statement
1.3
Hypothesis
1.4 Objective o f Research
1.5
Scope o f Research
1.6
Significance o f Study
1
1
3
5
5
5
6
LITERATURE REVIEW
2.1
Background o f F. Deltoidea
2.1.1
Phytochemistry and Pharmacology
2.1.2 Bloactlve Compounds o f Fi^us Deltoidea
2.1.2.1 Vltexln
2.1.2.2 Total Saponlns
7
7
8
9
10
11
1
2
2.1.2.3 Total Proteln
12
2.1.2.4 Total Polysaccharides
13
2.2 Extraction Process
14
2.2.1
Parameters Affecting the Extraction Process
15
2.2.1.1 Types o f Solvent
16
2.2.1.2 Extraction Temperature
16
2.2.1.3 Extraction Duration
17
2.2.1.4 Solvent to Solid Ratio
18
2.2.1.5 Particle Size
19
2.2.1.6 Agitation Speed
19
2.3
Extraction Process o f Ficus Deltoidea
20
2.4
Spray Drying Process in F. deltoidea Production
23
2.4.1
Phase in Spray Drying Process
25
2.4.2
Critical Parameters Affect Spray Drying
Process
28
2.4.2.1 Inlet Air Temperature
28
2.4.2.2 Feed Flow Rate
29
2.4.2.3 Atomization Pressure
30
2.4.2.4 Feed Temperature
30
2.4.2.5 Types o f Atomization Nozzle
31
2.4.2.6 Outlet Air Temperature
31
2.5
Optimization Process o f Ficus Deltoidea
32
2.5.1
Response Sufrace Methodology (RSM )
32
2.5.2 Application o f Response Surface Methodology
(RSM) on the Optimization of Extraction and
Spray Drying Processes
33
2.6
Summary
38
3
METHODOLOGY
39
3.1
Introduction
39
3.2 Raw Material
41
3.3
Chemicals and Reagents
41
3.4 Design of Experiment
42
3.5 Extraction Process
42
3.6.1
Total Solid Content
45
3.6.2 Vitexin Amount
46
3.6.3
Yield o f Extract
47
3.7
Spray Drying Process
47
3.8
Analytical M ethod for Spray Drying Process
51
3.8.1
Process Yield
51
3.8.2 Vitexin Amount
52
3.8.3
Moisture Content Analysis
52
3.9 Determination the Thermal Effect o f the Spray Drying
Temperature
53
3.9.1
The Thermal Effect o f Spray Drying
Temperature onto Biomarker Compounds in F.
deltoidea
54
3.9.1.1 Determination o f the Vitexin Amount
54
3.9.1.2 Determination o f Total Saponlns
55
3.9.1.3 Determination o f Total Protein
55
3.9.1.4 Determination o f Total Polysaccharides
57
3.10 Optimization Process
58
3.10.1 Analysis o f Variance (ANOVA)
58
3.10.2 F-dlstrlbutlon Test
59
3.10.3 Coefficient o f Multiple Determination (R2)
61
3.10.4 Response Surface Methodology
61
RESULTS AND DISCUSSIONS
63
4.1
Introduction
63
4.2 Optimization o f Extraction Process
64
4.3
Analysis o f Variance (ANOVA) for F. Deltoidea
Extraction Process
65
4.3.1
Null Hypothesis for the Polynomial Model for
Total Solid Content and Vitexin Amount in F.
Deltoidea Extract
66
4.3.2 Analysis o f Total Solid Content and Vitexin
Amount by Response Surface Methodology
(RSM)
68
4.4 Effect o f Extraction Temperature, Extraction Duration
and Solvent to Raw Material Ratio on Total Solid
Content
78
4.5
Effect o f Extraction Temperature, Extraction Duration
and Solvent to Raw Material Ratio on Vltexln Amount
81
4.6 Optimization o f F. Deltoidea Extraction using
Response Surface Methodology
85
4.7 Verification Phase: Confirmation o f Optimization
86
4.8
Optimization o f Spray Drying Process
88
4.9 Analysis o f Variance (ANOVA) for Spray Drying
Process
90
4.9.1
Null Hypothesis for the polynomial Model for
Process Yield, Vitexin Amount and Moisture
Content
90
4.9.2 Analysis o f Process Yield, Vltexln Amount and
Moisture Content by Response Surface
Methodology (RSM)
94
4.10 Effect o f Inlet Air Temperature, Feed Flow Rate, Air
Pressure and Feed Temperature on Process Yield of
Spray Drying
109
4.11 Effect o f Inlet Air Temperature, Feed Flow Rate, Air
Pressure and Feed Temperature on Amount o f Vltexln
during Spray Drying
116
4.12 Effect o f Inlet Air Temperature, Feed Flow Rate, Air
Pressure and Feed Temperature on Moisture Content of
Spray Drying
122
4.13 Optimization o f F. Deltoidea Spray Drying using
Response Surface Methodology
128
4.14 Verification Phase: Confirmation o f Optimization
130
4.15 Thermal Effect o f Spray Drying Process on the Quality
o f F. Deltoidea
131
4.16 Summary o f the Discussion
135
5
C O N C LU SIO N S
138
5.1
Conclusions
138
5.2 Recommendations
140
R E FE R E N C E S
141
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
The extraction condition o f Ficus deltoidea
21
2.2
Phases in spray drying Process
26
2.3
Application o f RSM for optimization o f extraction
process
34
2.4
Application o f RSM for optimization of spray drying
process
36
3.1
Processing parameters for the extraction o f F. deltoidea
43
3.2
Processing parameters and coded level for F. deltoidea
extraction process
44
3.3
Box Behnken Design for F. deltoidea extraction process
44
3.4
HPLC settings for the determination o f vitexin
47
3.5
The processing parameters for the spray drying o f F.
deltoidea
48
3.6
The processing parameters and coded level for spray
drying process
49
3.7
Box Behnken Design for spray drying process
50
3.8
The experimental design for the thermal effect in spray
drying process
54
3.9
Preparation o f reagents and standard for protein analysis
56
3.10
The analysis o f variance table for single factor
60
4.1
Box Behnken Design and response values for extraction
process
65
4.2
Analysis o f variance for total solid content in F. deltoidea
extract
66
4.3
Analysis o f variance for vitexin amount in F. deltoidea
extract
66
4.4
Summary o f ANOVA for total solid content from F.
4.5
Summary o f ANOVA for vitexin amount from F.
deltoidea extraction process
68
4.6
Coefficient estimate o f the quadratic model for total solid
content
69
4.7
Coefficient estimate o f the quadratic model for vitexin
amount
70
4.8
Experimental value and predicted value for total solid
content from the model
72
4.9
Experimental value and predicted value for vitexin
amount from the model
73
4.10
Optimum condition o f extraction process o f F. deltoidea
86
4.11
Comparison between predicted and experimental data at
the optimum condition
87
4.12
Box Behnken Design and response values for spray
drying process
89
4.13
Analysis o f variance for process yield o f F. deltoidea
spray drying process
91
4.14
Analysis o f variance for vitexin amount o f F. deltoidea
spray drying process
91
4.15
Analysis o f variance for moisture content o f F. deltoidea
spray drying process
91
4.16
Summary o f ANOVA for process yield from F. deltoidea
spray drying process
92
4.17
Summary o f ANOVA for vitexin amount from F.
deltoidea spray drying process
93
4.18
Summary o f ANOVA for moisture content from F.
deltoidea spray drying process
94
4.19
Coefficient estimate o f the quadratic model for process
yield
95
4.20
Coefficient estimate o f the quadratic model for vitexin
amount
97
4.21
Coefficient estimate o f the quadratic model for moisture
content
99
4.22
Experimental value and predicted value for process yield
from the model
101
4.23
Experimental value and predicted value for vitexin
amount from the model
102
4.24
Experimental value and predicted value for moisture
content from the model
103
4.26
Predicted values and modified values o f factor variables
at the optimum condition o f spray dry
130
4.27
Comparison between predicted and experimental data at
the optimum condition
131
4.28
Predicted values of experiment at each condition
132
4.29
Modified values o f experiment at each condition
132
4.30
Specification of freeze dried standardised water extract of
F. Deltoidea var. deltoidea (Abdullah et al., 2009)
133
4.31
Amount o f marker compounds before and after spray
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
2.2
2.3
2.4
3.1
3.2
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
Photo of Ficus deltoidea var. deltoidea
8
The chemical structure o f vitexin
10
General drying rate curve (Source: ^ellk and Wendel,
2005)
25
Flow chart o f spray drying process
26
Overall study flow chart
40
The inlet air temperature setting in Design Expert (V
6.0.8)
53
Predicted values versus actual values for total solid
content
74
Predicted values versus actual values for vitexin amount
74
Outlier T plot 17 experiments data points for total solid
content
75
Outlier T plots 17 experiments data points o f vitexin
amount
76
Normal-residual plots for total solid content
77
Normal-residual plots for vitexin amount
77
Interaction between extraction temperature and extraction
duration on total solid content
79
Interaction between extraction duration and solvent to
raw material ratio on total solid content
80
Interaction between extraction temperature and solvent to
raw material ratio on total solid content
81
Interaction between extraction temperature and extraction
duration on vitexin amount
83
Interaction between extraction duration and solvent to
raw material ratio on vitexin amount
84
Interaction between extraction temperature and solvent to
4.13
Predicted values versus actual values for process yield
104
4.14
Predicted values versus actual values for vitexin amount
105
4.15
Predicted values versus actual values for moisture content
105
4.16
Outlier T plot 29 experiments data points for process
yield
106
4.17
Outlier T plot 29 experiments data points for vitexin
amount
107
4.18
Outlier T plot 29 experiments data points for moisture
content
107
4.19
Normal-residual plots for process yield
108
4.20
Normal-residual plots for vitexin amount
108
4.21
Normal-residual plots for moisture content
109
4.22
Interaction between inlet air temperature and feed flow
rate on process yield
111
4.23
Interaction between feed flow rate and air pressure on
process yield
112
4.24
Interaction between inlet air temperature and air pressure
on process yield
113
4.25
Interaction between inlet air temperature and feed
temperature on process yield
114
4.26
Interaction between feed flow rate and feed temperature
on process yield
115
4.27
Interaction between air pressure and feed temperature on
process yield
115
4.28
Interaction between inlet air temperautre and feed flow
rate on vitexin amount
117
4.29
Interaction between feed flow rate and air pressure on
vitexin amount
118
4.30
Interaction between inlet air temperature and air pressure
on vitexin amount
119
4.31
Interaction between inlet air temperature and feed
temperature on vitexin amount
120
4.32
Interaction between feed flow rate and feed temperature
on vitexin amount
121
4.33
Interaction between air pressure and feed temperature on
vitexin amount
121
4.34
Interaction between inlet air temperature and feed flow
rate on moisture content
123
4.35
Interaction between feed flow rate and air pressure on
4.36
Interaction between inlet air temperature and air pressure
on moisture content
125
4.37
Interaction between inlet air temperature and feed
temperature on moisture content
126
4.38
Interaction between feed flow rate and feed temperature
on moisture content
127
4.39
Interaction between air pressure and feed temperature on
LIST OF ABBREVIATIONS
ANOVA
-
Analysis o f Variance
BBD
-
Box Behnken Design
C
-
Concentration
CCD
-
Central Composite Design
DF
-
Dilution factor
F
-
Fisher
F. deltoidea
-
Fi^us deltoidea
HPLC
-
High Performance Liquid Chromatography
MS
a-
Means o f squares for factor A
MS
e-
Means o f squares for error or residual
N
-
Normality
P
-
Probability
r s m
-
Response Surface Methodology
s s
-
Sum o f squares
SS
e-
Sum o f squares o f residual
SS
r-
Sum o f squares o f regression
SS
t-
Sum o f total squares
TLC
-
Thin Layer Chromatography
UV
-
Ulva Violet
UV-Vls
-
Ultraviolet-Visible
V
-
Volume
Var.
-
Varieties
LIST OF SYMBOLS
%
-
Percentage
°C
-
Degree Celsius
^g
-
Microgram
^l
-
Mlcrollter
^m
-
Micrometer
bo
-
Constant term
e
-
Residual
Fo
-
Calculated F value
g
-
Gram
H
a-
Alternative hypothesis
Ho
-
Null hypothesis
h
-
Hour
mg
-
Milligrams
min
-
Minutes
ml
-
Mililiter
mm
-
Milimeter
nm
-
Nanometer
ppm
-
Part per million
psi
-
Pounds per square inch
r\