MICROWAVE ASSISTED EXTRACTION OF CERIOPS DECANDRA SP. LEAVES FOR HYPOGLYCEMIC ACTIVE COMPOUNDS MUHAMMAD SYADDAD BIN ZAINOL A thesis submitted in fulfillment of the requirements for the award of the Degree of Bachelor of Engineering (Chemical) Faculty of Chemical Engineering & Natural Resources University Malaysia Pahang APRIL 2010
26
Embed
MICROWAVE ASSISTED EXTRACTION OF CERIOPS DECANDRA SP.umpir.ump.edu.my/3270/1/CD5855_SYADDAD_ZAINOL.pdf · MICROWAVE ASSISTED EXTRACTION OF CERIOPS DECANDRA SP. LEAVES FOR HYPOGLYCEMIC
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
MICROWAVE ASSISTED EXTRACTION OF CERIOPS DECANDRA SP.
LEAVES FOR HYPOGLYCEMIC ACTIVE COMPOUNDS
MUHAMMAD SYADDAD BIN ZAINOL
A thesis submitted in fulfillment
of the requirements for the award of the Degree of
Bachelor of Engineering (Chemical)
Faculty of Chemical Engineering & Natural Resources
University Malaysia Pahang
APRIL 2010
v
ABSTRACT
In this paper, a method of microwave-assisted extraction (MAE) was for the first
time used to extract total triterpenoid from Ceriops Decandra, which have been reported
to have hypoglycaemic activities towards human bodies. The extracts were directly
determined by colorimetric method without any further treatment. Compared with
Soxhlet extraction method and ultrasonic extraction method, MAE only need 20 min to
give the highest yield of triterpenoids at 1.1785%, while the Soxhlet extraction methods
need several hours and give lower yield. Several factors affecting the MAE extraction
rate were also discussed, such as extraction time, temperature and ratio of solvent to
material. Optimal conditions of MAE from this research can be concluded as follows: 20
min at 80 oC, the ratio of solvent to material is 25 by using 95% ethanol as the solvent.
vi
ABSTRAK
Dalam projek ini, pengekstrakan yang dibantu oleh gelombang mikro adalah
pertama kali digunakan untuk mengekstrak oleanolik asid yang merupakan ahli
kumpulan triterpenoid yang terdapat di dalam spesis bakau Ceriops Decandra, dimana
ia dikatakan dapat membantu menurunkan kadar gula di dalam darah manusia. Ekstrak
yang diperolehi dianalisis terus oleh teknik pengkoloran tanpa perlu melalui teknik
rawatan yang lain. Jika dibandingkan dengan teknik Soxhlet dan teknik Ultrasonik,
pengekstrakan yang dibantu oleh gelombang mikro hanya memerlukan 5 minit untuk
memperolehi hasil triterpenoid yang optimum iaitu pada kadar 1.1785% dan teknik yang
lain memerlukan masa yang lebih lama untuk memperolehi ekstrak dan hasil yang
diperolehi adalah lebih sedikit. Faktor-faktor yang mempengaruhi pengekstrakan yang
dibantu oleh gelombang mikro juga diambil kira termasuklah masa yang dilalui untuk
pengekstrakan berlangsung, suhu dan kadar pelarut terhadap bahan. Keadaaan optima
untuk, pengekstrakan yang dibantu oleh gelombang mikro adalah seperti berikut: 20
minit pada suhu 80 oC, kadar pelarut terhadap bahan adalah 25 dengan menggunakan
95% etanol sebagai bahan pelarut.
vii
TABLE OF CONTENTS
CHAPTER TITTLE PAGE
TITLE PAGE i
DECLARATION OF ORIGINALITY ii
AND EXCLUSIVENESS
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENT vii
LIST OF TABLE xi
LIST OF FIGURE xii
LIST OF ABBREVIATIONS xiv
1 INTRODUCTION
1.0 Introduction 1
1.1 Background of Study 1
1.2 Problem Statement 3
1.3 Objective 4
1.4 Scope of Research 4
1.5 Research Contribution 5
viii
2 LITERATURE REVIEW
2.1 Introduction 6
2.2 Introduction to Extraction 6
2.2.1 An Overview of Microwave Technology 7
2.2.2 Microwave energy – Electromagnetic Fields 10
2.2.3 Microwave Heating – Mathematical Aspects 11
2.2.4 MAE of Biological Active Compounds 14
2.3 Soxhlet Extraction 15
2.4 Colorimetric Analysis 17
2.4.1 Acid Hydrolysis 18
2.5 Triterpenoids 19
2.5.1 Structure and Classification 19
2.5.2 Biosynthesis of triterpenoids 20
2.5.3 Chemical Synthesis of Triterpenoids 22
2.5.4 Glycoside and Aglycone 22
2.5.5 Oleanolic Acid 23
2.6 Ceriops Decandra Species (Ceriops Decandra Sp.) 25
3 METHODOLOGY
3.1 Material Selection 27
3.1.1 Vanillin-Acetic Acid Solution 27
3.1.1.1 Vanillin 27
3.1.1.2 Acetic Acid 28
3.1.3 Perchloric Acid 28
3.1.4 Ethyl Acetate 29
3.1.5 Ethanol 29
3.2 Research Design 31
ix
3.3 Equipment and Apparatuses 32
3.3.1 List of Equipments 32
3.3.2 List of Apparatuses 32
3.4 Research Parameters (Single Factor Test) 33
3.4.1 Effect of Duration of Microwave Radiation 33
3.4.2 Effect of Ratio of Solvent to Material 33
3.4.3 Effect of Temperature 34
3.5 Experimental Stages 35
3.5.1 Literature Collection 36
3.5.2 Collection of Sample 36
3.5.3 Sample Preparation 36
3.5.4 Extraction of Triterpenoids 37
3.5.4.1 via Microwave Assisted Extraction 37
3.5.4.2 via Soxhlet Extraction 40
3.4.4 The Standard Curve 40
3.4.5 Determination of Total Triterpenoids 41
4 RESULT
4.1 Standard Curves and Determination of Total Triterpenoids 43
4.2
4.2.1 Result - Effect of Duration of Microwave Radiation 44
4.2.2 Result – Effect of Ratio of Solvent to Material 45
in MAE
4.2.3 Result – Effect of Temperature in MAE 46
4.2.4 Result – Effect of Duration in Soxhlet Extraction 47
5 CONCLUSION AND RECOMMENDATION
5.1 Conclusion 49
5.2 Recommendation 50
x
REFERENCES 51
APPENDIX 53
xi
LIST OF TABLES
TABLE TITLE PAGE
2.1 Comparison of Conversion Efficiencies of Various 9
Heating Source
2.2 Spectrophotometric Method for Organic Substance 18
2.3 Properties of Oleanolic Acid 25
xii
LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 The Ever Tightening Environmental Regulations 9
2.2 General Assembly of Soxhlet Extraction Equipment 17
2.3 Biosynthesis of Triterpenoids via MEP/DOXP Pathway 22
2.4 Chemical Arrangement of Oleanolic Acid 25
2.5 Fresh Ceriops Decandra Sp. Leaves Found at 26
Balok River, Pahang
3.1 Research Design for This Experiment 32
3.2 Experimental Stages 36
3.3 Satellite Views of Sample Collecting Area 37
3.4 (A) Sample and Solvent Loading Into Vessels 39
3.4 (B) Vessels Loading Into Sample Rotor 40
3.4 (C) Sample Rotor Loading Into Microwave Extarctor 41
xiii
3.5 Soxhlet Extraction in Progress 41
4.1 Standard Curve of Oleanolic Acid 45
4.2 Graph of Yield of Triterpenoids Vs Duration of 46
Microwave Radiation
4.3 Graph of Yield of Triterpenoids Vs Ratio of Solvent 47
to Material in Microwave Extraction
4.4 Graph of Yield of Triterpenoids Vs Temperature in 48
Microwave Extraction
4.5 Graph of Yield of Triterpenoids Vs Duration of Soxhlet 49
Extraction
xiv
LIST OF ABBREVIATIONS
P - Pressure
m - Mass
∆H - Enthalpy change of reaction
∆S - Entropy change of reaction
∆G - Energy change of reaction
T - Temperature
ρ - Density
μ - Viscosity of liquid (Pa.s)
h - Heat transfer coefficient
oC - Degree Celsius
kg - Kilogram
K - Degree Kelvin
m - Meter
n - Number of moles
L - Litre
1
CHAPTER 1
INTRODUCTION
1.0 Introduction
The design of environmentally friendly process for isolation of naturally
occurring triterpenoids via extraction and means of quantitative analysis of the yields
conceived for current research was among a few of its kind that is reported in current
literature. There have been a few publications along very close lines as discussed in the
literature survey. The field of triterpenoids is vast and there appears to be great potential
for inter-disciplinary systems research. The current research was carried out to
determine the optimum yield of triterpenoids from specific mangrove species under
certain condition which will be experimented. This chapter briefly introduces the
background of study, problem statement, objective, expected outcome, scope of research
and research contribution to the environment and society.
1.1 Background of Study
Mangrove trees from Rhizophoracae family are famous for its medicinal
purposes. Scientists around the world have conduct research and experiment on almost
all part of the mangrove from its root to the leaves on the sprouting branch and it have
been proven that each part of mangrove have its own distinctive medicinal properties.
Mangrove root, leaf and stem extracts have its own distinctive inhibitory properties that
can affect the growth of various human pathogenic organisms such as bacteria, fungi and
2
viruses (Hernandez and Perez, 1978). Gargles of mangrove bark can also cure throat
cancer (Garcia-Barriga, 1975). Leaf of Rhizophora apiculata polysaccharide extracts
said to blocked the expression of HIV-1 antigen in MT-4 cells and abolished the
production of HIV-1 p24 antigen in peripheral blood mononuclear cells (PBMC). It also
reduced the production of viral mRNA when added before virus adsorption. These
results suggest that Rhizophora apiculata Polysaccharide (RAP) may inhibit AIDS virus
in an early stage of its life cycle. It reported that extracts of Rhizophora mangle, which is
also known as red mangrove that is usually found in subtropical and tropical areas in
both hemispheres, extending to approximately 28°, had anti-diabetic and anti-
hyperglycemic property (Alarcon-Aguilara et al., 1998).
Recent research conduct on anti-hyperglycemic effect of the crude extract and
four fractions of Ceriops Tagal (Rhizophoracae family) were evaluated in normal
healthy sucrose-loaded and STZ-induced diabetic rats. The crude extract improved the
glucose tolerance of the sucrose-loaded rats significantly while in STZ-induced diabetic
rats the extract caused a fall in hyperglycemia. All of the four isolated fractions
improved the glucose tolerance of normal rats. Research conduct by Sur, Tapas Kumar
and his colleague (Sur et al, 2004) reveal that leaves of R.Apiculata extracts has
potential hypoglycemic action. Alcoholic extract of the leaves of this plant was prepared
and hypoglycemic activity was studied in fed rats, glucose loaded rats and streptozotocin
(STZ) induced diabetic rats.
Research conduct by V. Lakhsmi and her colleagues (V.Lakhsmi et al, 2006)
also reveals the hypoglycemic activity of the ethanolic extract of the roots of the
Rhizophora apiculata in rats (GLM and STZ models). On further fractionation of the
ethanolic extract into four fractions, the activity was localized in the chloroform and
aqueous fractions. These on purification led to the isolation of 7 pure compounds -