ULTRASONIC-ASSISTED EXTRACTION (UAE) OF TANNINS FROM STEM BARK OF JATROPHA CURCAS WAN ANIS BT WAN AZAMAN A thesis submitted in partial fulfillment of the requirements for the award of the degree of Bachelor of Chemical Engineering Faculty of Chemical and Natural Resources Engineering Universiti Malaysia Pahang NOVEMBER 2010
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ULTRASONIC-ASSISTED EXTRACTION (UAE) OF TANNINS FROM STEM
BARK OF JATROPHA CURCAS
WAN ANIS BT WAN AZAMAN
A thesis submitted in partial fulfillment of the
requirements for the award of the degree of
Bachelor of Chemical Engineering
Faculty of Chemical and Natural Resources Engineering
Universiti Malaysia Pahang
NOVEMBER 2010
v
ABSTRACT
Jatropha curcas, is gaining lot of importance in medicinal uses. The bark of
Jatropha Curcas presents a good source of functional compounds, such as
polyphenols. Ultrasound-assisted Extraction is evaluated as a simpler and more
effective to conventional extraction for isolation of tannins from Jatropha Curcas
stem bark. The purposes of this study are to extract tannins from stem bark of of
Jatropha Curcas and also to determine the optimum condition to extract the tannins.
The effects of solvent ratio (ethanol:water), temperature, extraction time, and
amplitude of sonication were studied. Ethanol was used as solvent in this experiment.
Extraction was done by using Ultrasounic-assisted extraction, which potentially
enhances extraction of polyphenolics. Purification of tannins using rotary evaporator
need to be done in order to obtained pure extract. Total phenolic content can be
determined spectrophotometrically using Folin–Ciocalteau method. To obtain the
amount of Gallic acid in extract, HPLC was used. In this experiment, the analysis
revealed that the optimized conditions were when the particle was 1.0 mm in size, a
temperature of 30⁰C, an amplitude of 40%, an extraction time of 30 minutes and 1:0
(v/v) ethanol:water ratio. For the conclusion, extraction of tannins using Ultrasound-
assisted Extraction was achieved and the optimum condition to extract tannins from
Jatropha Curcas stem bark was obtained.
vi
ABSTRAK
Pokok jarak pagar, mempunyai banyak kepentingan dalam perubatan. Kulit
pokok jarak pagar terdapat sumber berfungsi yang sangat baik, seperti polifenol.
Pengekstrakan Gelombang Bunyi Bantu dinilai sebagai lebih sederhana dan lebih
efektif untuk pengekstrakan konvensional untuk isolasi tanin dari kulit batang Jarak
Pagar. Tujuan dari penelitian ini adalah untuk mengekstrak tanin dari kulit batang
Jarak Pagar dan juga untuk menentukan keadaan optimum untuk mengekstrak tanin.
Pengaruh nisbah pelarut (etanol: air), suhu, waktu pengekstrakan, dan amplitud
sonikasi dipelajari. Etanol digunakan sebagai pelarut dalam percubaan ini.
Pengekstrakan dilakukan dengan menggunakan Pengekstrakan Gelombang Bunyi
Bantu, yang berpotensi meningkatkan pengekstrakan polifenol. Pengaslian tanin
menggunakan Pengwapan secara pusingan perlu dilakukan dalam rangka untuk
mendapatkan ekstrak yang asli. Jumlah fenolik kandungan dapat ditentukan secara
spektrofotometri menggunakan kaedah Folin-Ciocalteau. Untuk mendapatkan jumlah
asid Gaul dalam ekstrak, HPLC digunakan. Dalam kajian ini, analisis mendedahkan
bahawa keadaan optimum adalah ketika zarah adalah pada 1.0 mm dalam saiz, dengan
suhu 30 ⁰ C, amplitud 40%, waktu pengekstrakan selama 30 minit dan dengan nisbah
pelarut 1:00 (v / v) etanol: air. Sebagai kesimpulan, pengekstrakan tannin
menggunakan Pengekstrakan Gelombang Bunyi Bantu dan keadaan optimum untuk
mengekstrak tannin dari pokok jarak pagar diperolehi.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE PAGE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS AND SYMBOLS xii
LIST OF APPENDICES xiii
1 INTRODUCTION 1
1.1 Background of Study 1
1.2 Problem Statements 3
1.3 Objectives 4
1.4 Scopes of Study 4
1.5 Rationale & Significance 5
2 LITERATURE REVIEW 6
2.1 The Bark of Jatropha Curcas 6
2.2 Tannins 7
2.3 Medicinal Uses 8
viii
2.4 Ultrasound-assisted Extraction (UAE) 9
3 METHODOLOGY 11
3.1 Materials and Solvents 11
3.2 Apparatus 11
3.3 Standand Curve for UV-Vis Spectrophotometer
Preparation
3.3.1 Preparation of Gallic Acid Stock Solution
12
12
3.4 Preparation of Extracts 13
3.5 Sample Sizing 14
3.6 Ultrasound-assisted Extraction (UAE) 15
3.7 Rotary Evaporator 16
3.8 Chemical Analysis
3.8.1 Ultraviolet-Visible Spectroscopy (UV-Vis)
Analysis
3.8.2 High Performance Liquid Chromatography
(HPLC) Analysis
17
17
18
4 RESULT AND DISCUSSION 19
4.1 Result and Discussion
4.1.1 Standard Curve of Gallic acid by using
UV-Vis
4.1.2 Effect of Particle Size
4.1.3 Effect of Sonication Time
4.1.4 Effect of Amplitude of Extraction Yield
4.1.5 Effect of Extraction Temperature
4.1.6 Effect of Solvent Ratio
19
19
21
23
25
27
29
ix
5 CONCLUSION AND RECOMMENDATIONS 32
5.1 Conclusion 32
5.2 Recommendation 33
REFFERENCES 34
APPENDICES 38
x
LIST OF TABLES
TABLES NO TITLE PAGE
1.1 Uses of Jatropha Curcas 2
3.1 Preparation of Gallic acid Stock Solution 13
4.1 Concentration of total phenol content by using
UV-Vis
20
4.2 Percentage of yield extracted on effect of particle
size
21
4.3 Concentration of total phenol compounds on
effect of particle size
22
4.4 Effect of sonication time 23
4.5 Concentration of total phenol compounds on
effect of sonication time
24
4.6 Effect of amplitude of extraction 25
4.7 Concentration of total phenol compounds on
effect of amplitude
26
4.8 Effect of extraction temperature 27
4.9 Concentration of total phenol compounds on
effect of extraction temperature
28
4.10 Effect of solvent composition 29
4.11 Concentration of total phenol compounds on
effect of solvent ratio
31
xi
LIST OF FIGURES
FIGURES NO TITLE PAGE
2.1 Ultrasonic-assisted Extraction 10
3.1 Strip the bark from the stem. 14
3.2 Sieve Shaker 14
3.3 Sample extract in water bath by using UAE 15
3.4 Extract Filtration 16
3.5 Purification of total phenol by using rotary
evaporator.
16
4.1 Standard curves for Gallic acid 20
4.2 Graph on percentage of extraction yield of
particle size
22
4.3 Graph of effect of sonication time 24
4.4 Graph of effect of amplitude 26
4.5 Graph of effect of extraction temperature 28
4.6 Graph of effect of solvent ratio 30
xii
LIST OF ABBREVIATIONS AND SYMBOLS
Bhd. - Berhad
GAE - Gallic acid equivalent
HPLC - High-performance Liquid Chromatography
I.D - Inside Diameter
PVDF - Polyvinyledene Diflouride
PWE - Pressurized water extraction
TPC - Total phenol content
UAE - Ultrasonic-assisted Extraction
US - United State
SE - Soxhlet extraction
SFE - Supercritical fluid extraction
mg - miligram
mm - milimeter
mL - mililiter
min - minute
nm - nanometer
µg - Micron gram
v/v - Volume ratio
sp. - Species
s - Second
⁰C - Degree celcius
xiii
LIST OF APPENDICES
APPENDIX TITLE PAGE
A1 Gallic acid calibration curve 39
A2 Effect of Particle Size 40
A3 Effect of Sonication Time 41
A4 Effect of Amplitude of Extraction 42
A5 Effect of Temperature 43
A6 Effect of Solvent Ratio 44
A7 Bark of Jatropha Curcas (raw material) 45
1
CHAPTER 1
INTRODUCTION
1.1 Background of Study
Jatropha curcas (Linnaeus) is a multipurpose bush/small tree belonging to the
family of Euphorbiaceae. It is a plant with many attributes, multiple uses and
considerable potential. The plant can be used to prevent and/or control erosion, to
reclaim land, grown as a live fence, especially to contain or exclude farm animals and
be planted as a commercial crop. It is a native of tropical America, but now thrives in
many parts of the tropics and sub-tropics in Africa/Asia. It has few pests and diseases
and will grow under a wide range of rainfall regimes from 200 to over 1500 mm per
annum. In low rainfall areas and in prolonged rainless periods, the plant sheds its
leaves as a counter to drought (Openshaw, 1999).
Common names include Barbados Nut, Purging Nut, and Physic Nut. J. curcas
is a poisonous, semi-evergreen shrub or small tree, reaching a height of 6 m (20 ft)
(www.wikipedia.com). The genus name Jatropha derives from the Greek word jatros
(doctor) and trophe (food), which implies medicinal uses (Kumar et al., 2006).