THE EFFECT OF EXTRACTION PARAMETERS ON ANTIOXIDANT ACTIVITY OF Cosmos caudatus BADARIAH BT MOHD NASIR A thesis submitted to the Faculty of Chemical and Natural Resources Engineering in partial fulfillment of the requirements for the award of the Degree of Bachelor in Chemical Engineering (Biotechnology) Faculty of Chemical & Natural Resources Engineering Universiti Malaysia Pahang FEBRUARY 2013
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THE EFFECT OF EXTRACTION PARAMETERS ON ANTIOXIDANT
ACTIVITY OF Cosmos caudatus
BADARIAH BT MOHD NASIR
A thesis submitted to the Faculty of Chemical and Natural Resources Engineering in
partial fulfillment of the requirements for the award of the Degree of
Bachelor in Chemical Engineering (Biotechnology)
Faculty of Chemical & Natural Resources Engineering
Universiti Malaysia Pahang
FEBRUARY 2013
vi
THE EFFECT OF EXTRACTION PARAMETER ON
ANTIOXIDANT ACTIVITY OF Cosmos caudatus
ABSTRACT
The present of natural antioxidant in plant is well known and Cosmos
caudatus is one of the plants that have high content of antioxidant. The objective of
this study was to investigate the effect of operating conditions on extraction of total
antioxidant compound (TAC) from C. caudatus. TAC was extracted using 70 %
(v/v) acetone as the solvent for 4, 8 hour and 12 hours. C. caudatus was then
extracted at different to solid to solvent ratio of 1:20, 1:25 and 1:30 (w/v). Finally,
C. caudatus was extracted at different extraction temperature (60, 80 and 100 oC) for
30 min. The results show that the best extraction time to extract the TAC was after 8
hour extraction using solvent to sample ratio of 1:30 (w/v) and 66 oC of extraction
temperature. The TAC obtained was 28.8 mg AAE/g with the corresponding of
10.1% of its antioxidant activity. However, when considered the extraction
temperature, the highest value of TAC (19.5 mg AAE/g) was observed in extraction
at 80o C after 30 minute and for temperature 60 and 100
oC the TAC were 16.4 and
17.1 mg AAE/g. In HPLC analysis using the ratio sample to solvent at 8 hour, the
highest TAC was at 1:30 (2.368 mg AAE/g) and also for the extraction temperature
80 oC has highest TAC (1.84611 mg AAE/g). This trend was similar with the result
obtained from DPPH assay where 1:30 (w/v) and at 80 oC extraction has highest
TAC.
vii
KESAN PENGEKSTRATAN PARAMETER KE ATAS
ANTIOKSIDAN AKTIVITI Cosmos caudatus
ABSTRAK
Pada masa kini antioksida semula jadi dari tumbuhan diketahui umum dan
ulam raja merupakan salah satu tumbuhan yang mempunyai kandungan antioksida
yang tinggi. Objektif kajian ini adalah untuk mengkaji kesan pengekstratan
parameter ke atas antioksidan aktiviti Cosmos caudatus. Jumlah sebatian antioksidan
menggunakan 70 % aceton sebagai pelarut untuk 4, 8 dan 12 jam. Ulam raja
kemudiannye diekstrak pada nisbah pepejal kepada pelarut yang berbeza sebanyak
1:20, 1:25 dan 1:30 . Akhirnya ulam raja diekstrak pada suhu yang berbeza (60, 80
dan 100 oC) selama 30 minit. Keputusan menunjukkan bahawa pengekstrakan
terbaik untuk mengekstrak jumlah sebatian antioksidan adalah pada pasa 8 jam
menggunakan nisbah kepada pelarut 1:30 dan pada suhu 66 oC. Jumlah sebatian
antioksidan yg diperolehi adalah 28.8 mg AAE/g dengan bersamaan 10.1% aktiviti
antioksida. Walau bagaimanapun, apabila diekstark menggunakan suhu, nilai
tertinggi jumlah sebatian antioksida adalah 19.5 mg AAE/g diperhatikan pada suhu
80 oC selepas 30 minit. Dalam analisis HPLC menggunakan sampel nisbah pelarut
pada masa 8 jam pada setiap nisbah 1:20,1:25 dan 1:30, hasilnya 1:30 mempunyai
keputusan jumlah sebatian antioksida yang tertinngi iaitu 2.368 mg AAE/g dan juga
pada suhu 80 oC mempunyai nilai jumlah sebatian tertinggi iaitu 1.8461 mg AAE/g.
Hasil daripada analisis HPLC menunjukkan bacaan yang sama seperti analisis
menggunakan DPPH dimana 1:30 dan suhu 80 oC mempunyai nilai kandungan
antioksidan yang tinggi.
viii
TABLE OF CONTENTS
TITLE PAGE i
SUPERVISORS’S DECLARATION ii
STUDENT’S DECLARATION iii
DEDICATION iv
ACKNOWLEDGEMENT v
ABSTRACT vi
ABSTRAK vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SYMBOLS xiii
LIST OF ABBREVIATIONS xiv
CHAPTER 1 INTRODUCTION
1.1 Research Background 1
1.2 Problem Statement 3
1.3 Research Objective 3
1.4 Scope of Research 4
1.5 Significance of Study 5
CHAPTER 2 LITERATURE REVIEW
2.1 Cosmos caudatus 6
2.2 Extraction Parameter
2.2.1 Solid To Solvent Ratio
2.2.2 Effect Of Time On Extraction
2.2.3 Effect Of Temperature On Extraction
8
9
9
10
2.3 Review Related Research on Operating Parameter used for
Extraction.
12
2.4 Total Antioxidant 13
2.4 Equipment
2.5.1 Soxhlet extractor
2.5.2 Advantages and Disadvantages of Soxhlet Extractor
15
15
16
ix
2.5.3 rotary evaporator
2.5.4 UV Vis Spectrophotometer
2.5.5 High Performance Liquid Chromatography (HPLC)
17
18
19
CHAPTER 3 METHODOLOGY
3.1 Materials 21
3.2 Experimental procedure 21
3.3 Sample Preparation 22
3.4 Sample Extraction
3.4.1 Extraction Of C.caudatus Using Soxhlet Extraction Method
3.4.2 Solvent Extraction
24
24
26
3.5 Determination Antioxidant Activity of Cosmos caudatus extracts
using DPPH method
27
3.6 Determination of Ascorbic Acid Using High Performance Liquid
Chromatography (HPLC)
28
CHAPTER 4 RESULT AND DISCUSSION
4.1 Determination Calibration Curve of Total Antioxidant
Compounds
29
4.2 The Effect of Extraction Time on Antioxidant compounds from C.
caudatus
30
4.3 The Effect of Sample to Solvent Ratio On The Extraction Of
Antioxidant Activity From C. caudatus
33
4.4 The Effect of Temperature on The Extraction Of Antioxidant
Activity From C.caudatus
37
4.5 Analysis of Total Antioxidant Compound Using HPLC 40
CHAPTER 5 CONCLUSION AND RECOMENDATION
5.1 Conclusion 42
5.2 Recommendation 43
REFERENCE
APPENDICES
48
52
x
LIST OF TABLES
Pages
Table 2.1 The extraction operating condition used by
some researchers in determining the antioxidant
yield
12
Table 4.1 Data analysis for the effect of extraction time
on antioxidant activity in C. Caudatus
30
Table 4.2 The effect of sample to solvent (w/v) at
different time
34
Table 4.3 The analysis data for the effect of extraction
temperature
37
Table 4.4 The summary result for sample to solvent ratio
from C.caudatus by HPLC analysis
40
Table 4.5 The summary result for different extraction
temperature from C. caudatus by HPLC
analysis
41
Table A.1 The Absorbance Ascorbic Acid at Different
Concentration
52
xi
LIST OF FIGURES
Page
Figure 2.1 Soxhlet extractor 15
Figure 2.2 Theory UV-Vis 19
Figure 2.3 HPLC equipment 20
Figure 3.1 Experimental procedure 22
Figure 3.2 C.caudatus being weighted 23
Figure 3.3 Sample being dried using oven 23
Figure 3.4 Sample after grinder 23
Figure 3.5 Soxhlet apparatus 25
Figure 3.6 Rotary evaporator to separate solvent from sample 26