重陽木及火炭母草甲醇萃取物及其區分層之抗氧化性

Transcription

重陽木及火炭母草甲醇萃取物及其區分層之抗
氧化性
Antioxidant Activity of Methanol
Extracts and Subfractions from
Bischofia javanica and Polygonum
chinensis
研究生：劉宜珍（Yi-Chen Liu）
指導教授：李綉鈴（Prof. Shiow-Ling Lee）
大同大學
生物工程研究所
碩士論文
Thesis for Master of Science
Department of Bioengineering
Tatung University
中華民國九十七年七月
July 2008
誌謝
承蒙恩師李綉鈴教授於研究所期間給予生活、學業及實驗上的諄諄
教誨，使學生於研究路上得以成長茁壯，更於論文繕寫期間悉心校閱斧正，
方得以順利完成，特誌卷首。
文稿初成復蒙國立台灣大學食品科技研究所游若萩老師及本所官宜靜
老師詳加釜正，並惠與寶貴的建議，而使論文能更臻完善，在此獻上最深
的謝忱。
求學路途走來跌跌撞撞，感謝父母不斷的給予我安慰及支持，在我茫
然不知所措時，能很快找到自己的方向。感謝老姐及老哥的支持，妳們的
關懷及鼓勵是我最大的精神支柱，因為有你們，讓我能勇往直前並順利完
成學業。
同時，感謝實驗室的成員：力中、田甜及瑜芳學長姐、啟豪、宣百、
正豪、可凡、育坤及信瀚在各方面的幫忙。此外，更感謝我的球友小冠、
小觀、倫姐、龜爺、成哥與生工所的老師及同學，有你們的存在，讓我在
大同的日子增添許多動人的回憶。最後再次感謝曾經幫助過我的所有人。
往後人生路途上尚會遭遇許多困難，我會帶著老師們的教導與各位的
祝福，勇敢面對挑戰。
劉宜珍
謹誌於
大同大學生物工程研究所
中華民國九十七年八月
ABSTRCT
Bischofia javanica and Polygonum chinensis were extracted with boiling water,
methanol and ethyl acetate, respectively, and their antioxidant activity, contents of
total phenolic, flavonoids and condensed tannins were examined. The results
showed that methanol extract of B. javanica demonstrated the highest scavenging
activities against DPPH·, ABTS·+ and superoxide radicals with EC50 values of 50.4,
372.9 and 216.3 μg/ml, respectively. It was noteworthy that methanol extract
exhibited similar activity to ascorbic acid in DPPH· scavenging activity, and
significantly higher activity than ascorbic acid in superoxide radicals scavenging
activity under experimental condition. The highest ferric reducing antioxidant
power, ferrous ion chelating activity and inhibition of lipid peroxidation were also
found in methanol extract. Surprisingly, ferric ion chelating activity of methanol
extract was similar to those of ascorbic acid and BHA. Methanol extract of B.
javanica was further partitioned sequentially with n-hexane, diethyl ether, ethyl
acetate, and 1-butanol and then the antioxidant activity of each fraction was
determined. Ethyl acetate and water fractions possessed the highest DPPH·
scavenging activity with EC50 values of 48.5 μg/ml and 46.7 μg/ml, respectively,
which was similar to that of ascorbic acid. Ethyl acetate fraction obtained the
highest ABTS·+ and superoxide anion radical scavenging activity as well as FRAP.
It was noted that EC50 values of ABTS·+ scavenging activity of ethyl acetate fraction
was 219.4 μg/ml, similar to those of BHA and ascorbic acid. Nevertheless,
1-butanol fraction possessed the highest activity in ferrous ion-chelating activity and
inhibition of lipid peroxidation. The correlation coefficient from Pearson correlation
analysis indicated a positive relation between antioxidant activity and contents of
total phenolics and condensed tannins. However, correlation of antioxidant activity
I
with content of flavonoids was not found.
Among three extracts, methanol extract of P. chinensis displayed the highest
scavenging activities against DPPH·, ABTS·+ and superoxide radicals, FRAP,
ferrous ion chelating activity, and inhibition of lipid peroxidation. It was noted that
ferrous ion chelating activity in methanol extract with 55.9 μmol EDTA equivalents
/ g was similar to those of ascorbic acid and BHA. Moreover, superoxide anions
radicals scavenging activity with EC50 values of 232.5 μg/ml was significantly
higher than that of ascorbic acid. Methanol extract of P. chinensis was further
partitioned sequentially with n-hexane, diethyl ether, ethyl acetate and 1-butanol and
then the antioxidant activity of each fraction was determined. Ethyl acetate fraction
of P. chinensis methanol extract exhibited the highest activities in DPPH·, ABTS·+
and superoxide radicals scavenging activities, and reducing power, but the highest
ferrous ion-chelating activity and inhibition of lipid peroxidation activity was found
in diethyl ether fraction. The DPPH· scavenging activity of ethyl acetate fraction
and the ferrous ion-chelating activity of diethyl ether fraction were similar to those
of ascorbic acid and BHA, but the superoxide anions radicals scavenging activities
of all five fraction were significantly higher than that of ascorbic acid. The contents
of total phenolics, flavonoids and condensed tannins gave the strong positive
correlations with antioxidant activities by Pearson correlation analysis.
II
摘要
本研究分別以熱水、甲醇及乙酸乙酯萃取重陽木及火炭母草，並測定萃取
物之抗氧化能力及其總酚、黃酮與縮合單寧之含量。結果顯示重陽木之甲醇萃
取物具最高DPPH、ABTS與超氧陰離子自由基清除能力，其EC50 分別為 50.4
μg/ml、372.9 μg/ml及216.3 μg/ml。值得注意是其DPPH自由基清除能力與抗壞
血酸相似，而其超氧陰離子自由基清除能力則遠高於抗壞血酸。甲醇萃取物亦
具最高之還原力、螯合亞鐵離子能力及脂質過氧化抑制能力，其螯合亞鐵離子
能力與抗壞血酸及BHA相似。進一步將重陽木甲醇萃取物經正己烷、乙醚、乙
酸乙酯及正丁醇進行區分後，並評估五區分層之抗氧化能力，其中DPPH自由
基清除能力以乙酸乙酯區分層及水層最佳，其 EC50 分別為 48.5 μg/ml 與
46.7μg/ml，與抗壞血酸相似。乙酸乙酯區分層亦具有最高之ABTS自由基清除
能力及超氧陰離子自由基清除能力與還原力，其中ABTS自由基清除能力與抗
壞血酸及BHA相似，其EC50為219.4 μg/ml。正丁醇區分層則是具最高螯合亞鐵
離子能力及脂質過氧化抑制能力。經皮爾森相關分析發現此抗氧化能力與總酚
與縮合單寧含量呈高度正相關，但與黃酮含量並無顯著相關。
火炭母草三種萃取物中，以甲醇萃取物具最高之DPPH、ABTS、超氧陰離
子自由基清除能力、還原力、螯合亞鐵離子能力及脂質過氧化抑制能力。其中
螯合亞鐵離子能力約為55.9 μmol EDTA / g，與抗壞血酸及BHA相似。而，超
氧陰離子自由基清除能力則顯著高於抗壞血酸，其EC50約為232.5 μg/ml。火炭
母草甲醇萃取物經過正己烷、乙醚、乙酸乙酯及正丁醇進行區分並測定各區分
層之其抗氧化能力，發現乙酸乙酯區分層具有最佳之DPPH、ABTS、超氧陰離
子自由基清除能力、還原力，而螯合亞鐵離子能力及脂質過氧化抑制能力則以
乙醚區分層最佳。乙酸乙酯區分層之 DPPH自由基清除能力及乙醚區分層之螯
合亞鐵離子能力均與抗壞血酸及BHA相似，而五區分層之超氧陰離子自由基清
除能力均比抗壞血酸高。經皮爾森相關分析發現此抗氧化能力與總酚、黃酮與
III
縮合單寧含量呈高度正相關。
IV
TABLE OF CONTENTS
ABSTRACT………………………..…..………………………………………......... I
ABSTRACT IN CHINESE………….. …………………………............................. III
TABLE OF CONTENTS…………………..…………………………………….. .. V
LIST OF FIGURES…………………..…………………………………………..... VIII
LIST OF TABLES…………………..……………………………………………... XII
CHAPTER 1 INTRODUCTION…………………..……………………………... 1
CHAPTER 2 BACKGROUND…………………..……………………………..… 2
2.1 Medicinal plants…………………..……………………………………..…… 2
2.1.1 Bischofia javanica…………………..……………………………..….... 2
2.1.2 Polygonum chinensis Linn. …………………..……………………..….. 5
2.2 Injury that free radical produces to the human body…………………..…..…. 5
2.2.1 Definition of free radical…………………..…………………………..… 5
2.2.2 Reactive oxygen and reactive nitrogen species…………………..…..….. 7
2.2.3 Sources of ROS and RNS…………………..…………………………..... 8
2.2.4 Oxidative or nitrosative stress damage of free radical…………………... 8
2.2.4.1 Oxidative or nitrosative stress damage to biomolecules………..... 11
2.2.4.1.1 Oxidative or nitrosative damage to deoxyribonucleic
acid…………………..………………………….…….... 11
2.2.4.1.2 Lipid…………………………………………………...… 14
2.2.4.1.3 Protein…………..………………………………………. 14
2.2.4.2. Oxidative or nitrosative stress and disease………………..…..…. 18
2.2.4.2.1 Diabetes…………………..………………………..…… 18
2.2.4.2.2 Neurodegenerative disorders…………………..…..…… 20
2.2.4.2.3 Cardiovascular disease…………………..………..……. 24
2.2.4.2.4 Cancer…………………..………………………..…….. 24
2.3 Antioxidant…………………..…………………………………………..…… 26
2.3.1 Antioxidant mechanisms…………………..……………………..……… 26
2.3.1.1 Preventive antioxidants…………………..……………………..... 28
2.3.1.1.1 Transient metal chelators…………………..………….... 28
2.3.1.1.2 Singlet oxygen quenchers…………………..……..…… 28
2.3.2.1 Chain breaking antioxidants…………………..……………..…… 30
2.3.2 Antioxidant defense in vivo…………………..……………………..…… 31
2.3.3.1 Antioxidant enzymes…………………..……………………..…... 31
2.3.3 Non-enzymes antioxidant…………………..………………………..…... 31
2.3.3.1 Vitamin C…………………..…………………………………..… 35
2.3.3.2 Vitamin E…………………..…………………………………..…. 35
V
2.3.3.3 Carotenoids…………………..…………………………………... 38
2.3.3.4 Thiol antioxidants — glutathione and lipoic acid………………... 38
2.3.3.5 Phnolic compounds…………………..………………..…………. 41
CHAPTER 3 MATERIALS AND METHODS………………….. ..…………….. 50
3.1 Experimental procedures…………………..………………………………….. 50
3.2 Tested herbal medicines…………………..……………………………..…… 51
3.3 Chemicals…………………..……………………………………………..….. 51
3.4 Preparation of extracts from herb…………………..……………………..….. 52
3.4.1 Boiling water extraction of herb…………………..…………………..…. 52
3.4.2 Organic solvents extraction of herb…………………..………………...... 52
3.5 Determination of antioxidant activities…………………..………………….... 53
3.5.1 Radical-scavenging activity…………………..………………………......
3.5.1.1 DPPH radical scavenging activity…………………..………….....
3.5.1.2 ABTS cation radical scavenging activity...…………………..……
3.5.1.3 Superoxide anion radical scavenging activity………………….....
3.5.2 Ferric reducing antioxidant power………..……………………………....
3.5.3 Ferrous ion-chelating activity……………………..……………………...
3.5.4 Inhibition of lipid peroxidation assay………………………………….....
3.6 Contents of antioxidant components…………………………..………………
3.6.1 Contents of total phenolic……………………………………..………….
3.6.2 Contents of flavonoids……………………………………………………
3.6.3 Content of condensed tannins………………………………………..…...
3.7 Preparation of extracts in organic solvents from crude extracts of methanol…
3.8 Statistical analysis…………………..………………….……………..……….
53
53
55
57
59
61
62
64
64
65
65
66
66
67
67
67
67
67
73
75
76
80
82
CHAPTER 4 RESULTS AND DISCUSSION………………..………..…………
4.1 Bischofia javanica…………………..……………………………..………….
4.1.1 Extraction yields from different solvents………………….. ..…………...
4.1.2. Antioxidant activities of various extracts from B. javanica…..…………
4.1.2.1 Radical-scavenging activity………………….………..…………..
4.1.2.2 Ferric reducing antioxidant power………..…………..…………...
4.1.2.3 Ferrous ion-chelating activity…………………..…..……………..
4.1.2.4 Inhibition of lipid peroxidation…………………..……………….
4.1.3 Contents of antioxidant components………………………………..……
4.1.4 Antioxidant activity of different solvents of B. javanica………………...
4.1.5Antioxidant activity of subfraction from methanol extract of B.
javanica……………….…………………………………………..…….. 82
4.1.5.1 Yield of subfraction derived from B. javanica methanol
extract……………………..…………………..……………..…… 82
VI
4.1.5.2 Radical-scavenging activity……………..….……………..……… 82
4.1.5.3 Ferric reducing antioxidant power………..………………..……... 88
4.1.5.4 Ferrous ion-chelating activity……………..………………..…….. 88
4.1.5.5 Inhibition of lipid peroxidation…………….……………………... 90
4.1.5.6 Contents of ingredients subfractions of methanol extract form B.
javanica……………………………………………………………. 94
4.1.5.7 Correlations of antioxidant activities with contents of antioxidant
compounds………………………………………………....……… 97
4.1.6. Conclusion…………………..………………………………..…………. 99
4.2 Polygonum chinensis Linn…………………..……………………..…………. 100
4.2.1 Extraction yields from different solvents………………….. ..…………... 100
4.2.2. Antioxidant activities of various extracts from P. chinensis…………..… 102
4.2.2.1 Radical-scavenging activity…………………..……………..…… 102
4.2.2.2 Ferric reducing antioxidant power………..………………..…..… 106
4.2.2.3 Ferrous ion-chelating activity…………………..………..….…… 106
4.2.2.4 Inhibition of lipid peroxidation…………………..…..…………... 109
4.2.3 Contents of antioxidant components…………………..…..…………….. 109
4.2.4 Antioxidant activity of different solvents of P. chinensis ………..………. 113
4.2.5 Antioxidant activity of subfraction from methanol extract of P.
chinensis……. …………………..……………………………………....... 113
4.2.5.1 Yield of subfraction derived from P. chinensis methanol extract…. 113
4.2.5.2 Radical-scavenging activity…………………..…..………..……… 113
4.2.5.3 Ferric reducing antioxidant power………..……………..………... 120
4.2.5.4 Ferrous ion-chelating activity………………………..……..…….. 120
4.2.5.5 Inhibition of lipid peroxidation……………………..……………. 120
4.2.5.6 Contents of ingredients subfractions of methanol extract form P.
chinensis…………………………………..…...………………….. 123
compounds………………………………………………………... 127
4.2.6. Conclusion…………………………………………………..…………... 131
CHAPTER 5 REFERENCES..……………………………………………………. 132
CHAPTER 6 APPENDIXES……………………………………..………………... 144
VII
LIST OF FIGURES
Figure 2.1
The structures of tannins isolated from B. javanica. Bischofianin (1),
geraniin (2), corilagin (3), furosin (4), punicalagin (5), procyanidin B-1
(6), phenazine derivative (la), phenazine bislactone (1b),
1,2,3,6-tetra-O-galloyl-β-D-glucopyranose (1c), and phillylaeoidin E
(1d).…………………………………………………………………..….. 3
Figure 2.2
The structures of betulinic acid (1) and its derivatives, betulonic acid (2),
3β-O-(Z)-coumaroylbetulinic acid (3), and 3β-O-(E)-coumaroylbetulinic
acid (4)..………………………………………………………………..… 4
Figure 2.3
Chemical structures of isolates from Polygonum chinensis.
Stigmast-4-ene-3,6-dione (1), stigmastane-3,6-dione (2), hecogenin (3),
25R-spirost-4-ene-3,12-dione
(4),and
aurantiamide
acetate
(5)..…........................................................................................................... 6
Figure 2.4
Exogenous and endogenous sources of ROS and RNS.……………..….... 10
Figure 2.5
Reaction of guanine with hydroxyl radical………………………………. 13
Figure 2.6
The overall process of lipid peroxidation.…………………….. ………… 15
Figure 2.7
Various pathways of lipid peroxidation………………………..………... 16
Figure 2.8
Oxidative or nitrosative modifications of protein amino acids.…..……... 17
Figure 2.9
Hyperglycemia in an organism stimulates ROS or RNS formation from a
variety of sources……………………………………………………….... 21
Figure 2.10 Mechanism of oxidative stress in neurodegenerative disorders…….. .…. 23
Figure 2.11 The pathways of ROS/RNS generation in cardiovascular system…....….. 25
Figure 2.12 Three stages model of carcinogenesis…………………………..…..……. 27
Figure 2.13 Antioxidant fates of nitric oxide on low density lipoprotein oxidation. .... 32
VIII
Figure 2.14 Relevant relationships between free radical and antioxidant………….…. 33
Figure 2.15 Antioxidants enzymic (A) Intracellular and extracellular distribution, (B)
Enzymatic degradation of reactive oxygen species……………………… 34
Figure 2.16 Ascorbic acid functions. (A) Various forms of ascorbic acid and its
reaction with radicals (R•). (B) Ascorbate-glutathione antioxidant
systems…………………………………………………………………… 36
Figure 2.17 (A) Chemical structure of Vitamin E. (B) Antioxidation mechanism of
α-tocoferol………………………………………………………….......... 37
Figure 2.18 Structure of carotenoids………………………………………………….. 39
Figure 2.19 Structures of of reduced (GSH) and oxidizes (GSSG) glutathinone…...... 40
Figure 2.20 Role of GSH in oxidation of protein sulphydry groups………………….. 42
Figure 2.21 Relationship of GSH and other antioxidants…………………………….. 43
Figure 2.22 Structure of lipoic acid and dihydrolipoic acid………………………….. 44
Figure 2.23 Skeletal structure of flavonoid and related structures…………………… 46
Figure 2.24 Flavonoids can exercise their antioxidant activity in several ways. (A)
Activity was attributed to their hydrogen-donating ability. (B) Metallic
ion complexation by flavonoids via the 30-40-o-diphenolic group in the
B ring (a) and ketol structures 4-keto, 3-hydroxy in the C ring (b) or
4-keto, 5-hydroxy in the C and A rings (c)…………………………….… 47
Figure 2.25 Typical tannins (polyphenolics). 1 is a typical procyanidin or condensed
tannin (PC), made up of catechin and epicatechin. 2 is a
hydrolyzable tannin (polygalloyl glucose), made up of a glucose core
esterified with gallic acid residues. 3 is a phlorotannin (tetrafuhalol A),
made up of phloroglucinol subunits……………………………………… 49
Figure 3.1
Experimental procedures…………………………………………….…... 50
IX
Figure 3.2
Scheme for Scavenging the DPPH Radical by an antioxidant. RH,
antioxidant…………………………………………………………….….. 54
Figure 3.3
Scheme for scavenging the ABTS·+ by an antioxidant……....................... 56
Figure 3.4
Scheme for superoxide anion was generated in a PMS- NADH system.... 58
Figure 3.5
Scheme for Fe (III) (TPTZ) 2Cl3 and its reduction by an antioxidant……. 60
Figure 3.6
The reaction of inhibition lipid peroxidation assay……………………… 63
Figure 4.1
Radical scavenging activities of different extracts of B. javanica.●,
Boiling water； ○, Methanol； ▼, Ethyl acetate；△, Ascorbic acid；■,
BHA………………………………………….…………………………… 70
Figure 4.2
Inhibition of lipid peroxidation of boiling water and methanol extracts
from B. javanica and BHA………………………………………………. 78
Figure 4.3
Radical scavenging activity of derived fractions from methanol extract of
B. javanica. ●, Methanol extract； ○,n-Hexane fraction； ▼, Diethyl
ether fraction；△, Ethyl acetate fraction； ■, 1-Butanol fraction；□,
Water fraction；◆, Ascorbic acid；◇, BHA…………………………….. 86
Figure 4.4
Inhibition of lipid peroxidation activity of derived fractions from
methanol extract of B. javanica by TBARS method…………………….. 92
Figure 4.5
Radical scavenging activities of different extracts of P. chinensis.●,
Boiling water； ○, Methanol； ▼, Ethyl acetate；△, Ascorbic acid；■,
BHA. ………………………………………………………………….….. 103
Figure 4.6
Inhibition of lipid peroxidation of boiling water and methanol extracts
from P. chinensis and BHA………………………………………………. 110
Figure 4.7
Radical scavenging activity of derived fractions from methanol extract of
P. chinensis. ●, Methanol extract； ○,n-Hexane fraction； ▼, Diethyl
ether fraction；△, Ethyl acetate fraction； ■, 1-Butanol fraction；□,
Water fraction；◆, Ascorbic acid；◇, BHA.…………………………….. 116
X
Figure 4.8
Inhibition of lipid peroxidation activity of derived fractions from
methanol extract of P. chinensis by TBARS method…………………….. 124
Figure 6.1
Standard curve of DDPH ·. (A) Calibration curve of ascorbic acid
equivalents. (B) Calibration curve of trolox equivalents………………… 144
Figure 6.2
Standard curve of ABTS ·+. (A) Calibration curve of ascorbic acid
equivalents. (B) Calibration curve of trolox equivalents………………… 145
Figure 6.3
Standard curve of superoxide anion radical. (A) Calibration curve of
ascorbic acid equivalents. (B) Calibration curve of trolox equivalents….. 146
Figure 6.4
Standard curve of FRAP methol. (A) Calibration curve of FeSO4 147
equivalents. (B) Calibration curve of trolox equivalents…………………
Figure 6.5
Standard curve of EDTA equivalents…………………………………
148
Figure 6.6
Standard curve of gallic acid equivalents………………………………
149
Figure 6.7
Standard curve of quecetin equivalents………………………………..
150
Figure 6.8
Standard curve of catechin equivalents………………………………...
151
XI
LIST OF TABLES
Table 2.1 Characteristic and features of ROS and RNS……....……………………... 9
Table 2.2 Potential markers of oxidative or nitrosative damage in vivo……………... 12
Table 2.3 Biomarkers of oxidative/ nitrosative damage associated with some human
disease……………………………………………………………………... 19
Table 2.4 Potential causal factors leading to oxidative stress in various
neurodegenerative disorders……………………………………………….. 22
Table 2.5 Preventive antioxidants and chain breaking antioxidants………………..... 29
Table 4.1 Extraction yield of B. javanica from different solvents…………………… 68
Table 4.2 Radical scavenging activity of various extracts from B. javanica
extracts…………………………………………………………………….. 72
Table 4.3 Reducing activity of B. javanica extracts using FRAP method…………… 74
Table 4.4 Ferrous ion-chelating capacity of various extracts from B. javanica……… 77
Table 4.5 Inhibition of lipid peroxidation activity of various extracts from B.
javanica……………………………………………………………………. 79
Table 4.6 Contents of total phenolics, flavonoids, and condensed tannins in various
extracts from B. javanica………………………………………………….. 81
Table 4.7 Antioxidant activity and ingredients contents of various solvent extracts
from B. javanica……………………………………….…………………... 83
Table 4.8 Yields of derived fractions from methanol extract of B. javanica…………. 84
Table 4.9 Radical scavenging activity of derived fractions from methanol extract of
B. javanica…………………………………………………………………. 87
XII
Table 4.10 Reducing activity of derived fractions from methanol extract of B.
javanica by FRAP method…………………………………………………. 89
Table 4.11 Ferrous ion-chelating capacity of derived fractions from methanol extract
of B. javanica………………………………………………………………. 91
Table 4.12 Inhibition of lipid peroxidation activity of derived fractions from methanol
extract of B. javanica………………………………….…………………… 93
Table 4.13 Total phenolics, flavonoids, and condensation tannins of derived fractions
from methanol extract of B. javanica……………………………………… 95
Table 4.14 Antioxidant capacity and ingredients contents of derived fractions from
methanol extract of B. javanica……………………………………………. 96
Table 4.15 Correlation coefficients between assays in fractions of methanol extract in
B. javania……………………………………………………………........... 98
Table 4.16 Extraction yield of P. chinensis extracts from different solvents. ……….
101
Table 4.17 Radical scavenging activity of various extracts from P. chinensis extracts.. 105
Table 4.18 Reducing activity of P. chinensis extracts using FRAP method…………… 107
Table 4.19 Ferrous ion-chelating capacity of various extracts from P. chinensis…..… 108
Table 4.20 Inhibition of lipid peroxidation activity of various extracts from P.
chinensis……………………………………………………………………. 111
Table 4.21 Contents of total phenolics, flavonoids, and condensed tannin in various
extracts from P. chinensis………………………………………………....... 112
Table 4.22 Antioxidant activity and ingredients contents of various solvent extracts
from P. chinensis…………………………………………………………. 114
Table 4.23 Yields of derived fractions from methanol extract of P. chinensis………. 115
Table 4.24 Radical scavenging activity of derived fractions from methanol extract of
XIII
P. chinensis……..………………………………………………………….. 118
Table 4.25 Reducing activity of derived fractions from methanol extract of P.
chinensis by FRAP method……………………………………………....... 121
of P. chinensis……………………………………………………………... 122
extract of P. chinensis…………………………………………
125
Table 4.28 Total phenolics, flavonoids, and condensation tannins of derived fractions
from methanol extract of P. chinensis…………………………………… 126
Table 4.29 Antioxidant capacity and ingredients contents of derived fractions from
methanol extract of P. chinensis…………………………………………. 128
Table 4.30 Correlation coefficients between assays in fractions of methanol extract in
P. chinensis………………………………………………………………. 129
XIV
CHAPTER 1 INTRODUCTION
The use of Traditional Chinese Medicine (TCM) for health reasons started
thousands of years ago and Asian people still prefer to combine herbal medicines
with western treatment, especially in China. The global market of TCM was
growing at 10-20% annually. In 1987, the World Health Organization (WHO)
emphasized the importance of scientific investigations into indigenous herbal
supplements. More evidence showed that herbal supplements possessed antioxidant
effects. TCM couled reduce the acute or chronic disease development on the
oxidative stress (Zhu et al., 2004).
Cheng (2004) evaluated boiling water extracts of 31 herbal medicines for their
antioxidant activities, and revealed that Bischofia javanica, Mirabilis Jalapa,
Chamaesyce thymifolia, Mallotus paniculatus, Mimosa pudica, and Polygonum
chinensis exhibited greater antioxidant activities.
In the present study, antioxidant properties and phytochemical charactenistics
of extracts from B. javanica and P. chinensis, as well as fractions derived from
methanol extract of herbs were determined.
1
CHAPTER 2 BACKGROUND
2.1 Medicinal plants
2.1.1 Bischofia javanica
Bischofia javanica (Euphorbiaceae) was a common subtropical tree and
cultivated as an avenue tree. It was as a tonic for babies, as a diuretic, and for the
topical treatment of ulcers, sores and boils; anthelmintic and antidysenteric activities
(Khan et al., 2001). Tanaka et al. (1995) isolated active component from B. javanica
inducding dimeric ellagitannin, bischofianin, and other tannins compound as shown
in Fig. 2.1. The 70% ethanol extract of the bark of B. javanica, exhibited moderate
inhibition in prostaglandin biosynthesis and inflammation activity (Dunstan et al.,
1997). Extract of B. javanica leave prepared with methanol and ethyl acetate
inhibited the growth of Bursaphelenchus xylophilus, some Gram positive (Bacillus
cereus, Bacillus coagulans, Bacillus megaterium, Bacillus subtilis, Lactobacillus
casei,
Micrococcus
luteus,
Micrococcus
roseus,
Staphylococcus
albus,
Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus faecalis) and
some
Gram
negative
(Agrobacterium
tumefaciens,
Citrobacter
freundii,
Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Neisseria
gonorrhoeae, Proteus mirabilis, Proteus ulgaris ,Pseudomonas aeruginosa,
Salmonella typhi, Salmonella typhimurium and Serratia marcescens) (Alen et al.,
2000; Khan et al., 2001). Betulinic acid and it’s derivatives isolated from the CHCl3
extract of the bark of B. javanica were found to be catalytic inhibitors of DNA
topoisomerase II activitiy with IC50 values ranging from 0.38 to 56.12 μM (Wada &
Tanaka, 2005). The structures of betulinic acid and its derivatives were shown in Fig.
2.2.
2
Fig. 2.1 The structures of tannins isolated from B. javanica. Bischofianin (1), geraniin (2), corilagin (3), furosin (4), punicalagin (5), procyanidin
B-1 (6), phenazine derivative (la), phenazine bislactone (1b), 1,2,3,6-tetra-O-galloyl-β-D-glucopyranose (1c), and phillylaeoidin E (1d).
(Tanaka et al., 1995)
3
Fig. 2.2 The structures of betulinic acid (1) and its derivatives, betulonic acid (2),
3β-O-(Z)-coumaroylbetulinic acid (3), and 3β-O-(E)-coumaroylbetulinic
acid (4).
(Wada & Tanaka, 2005)
4
2.1.2 Polygonum chinensis Linn
Polygonum chinensis Linn (Polygonaceae) was used in its entirety as a
folk medicine in Taiwan to treat many infectious diseases such as inflammation,
diphtheria, and dandy fever (Huang et al., 2008; Tsai et al.,1998).
Tsai et al. (1998) isolated anti-inflammation and anti-allergic components
from P. chinensis such as 25R-spirost-4-ene-3, 12-dione, stigmast-4-ene-3,
6-dione, stigmastane-3, 6-dione, hecogenin and aurantiamide acetate (Fig. 2.3).
2.2 Damage that free radical produces to the human body
Oxygen, while undeniable essential for life was involved in the destruction
of tissue and causes failure of function. Oxygen-free radicals (OFR), more
generally known as reactive oxygen species (ROS) as well as reactive nitrogen
species (RNS) were products of normal cellular metabolism. They were
recognized for playing twin roles as both harmful and useful. Useful effects of
them occurred at low or moderate concentrations and involved physiological
roles in intracellular signaling systems, and redox regulation systems. On the
contrary, at high concentrations, they can be important mediators of damage to
various biomolecules including proteins, lipids as well as lipoproteins, and DNA
involved in age-dependent diseases such as cancer, arteriosclerosis, arthritis, and
neurodegenerative disorders (Ridnour et al., 2005; Valko et al., 2004; Valko et
al., 2005; Valko et al., 2006).
2.2.1 Definition of free radical
Free radicals were be defined as molecules or molecular fragments
containing one or more unpaired electrons in atomic or molecular orbital. The
5
Fig.2.3.
Chemical structures of isolates from Polygonum chinensis.
Stigmast-4-ene-3,6-dione (1), stigmastane-3,6-dione (2), hecogenin (3),
25R-spirost-4-ene-3,12-dione (4),and aurantiamide acetate (5).
(Tsai et al., 1998)
6
unpaired electrons had a considerable degree of reactivity. They could readily
react with most bio-molecules, starting a chain reaction of free radical formation
(Liochev & Fridovich, 1994; Valko et al., 2004; Winterbourn, 1993).
2.2.2 Reactive oxygen and reactive nitrogen species
Reactive radical species included a wide range of oxygen-, carbon-, sulfurradicals, originating from the superoxide radical, hydrogen peroxide, and lipid
peroxides. There could be formed by chelates of amino-acids, peptides, and
proteins with the toxic metals (Shchepinov, 2007; Valko et al., 2006).
Radicals derived from oxygen or nitrogen represented the most important
class of radical species generated in living systems (Valko et al., 2007; Valko et
al., 2006). Reactive oxygen species could be oxygen centered. Molecular
oxygen (dioxygen) had a unique electronic configuration and was itself a radical.
ROS, such as primary superoxide (O2•-), hydroxyl (•OH), carbonate (CO3•-),
peroxyl (RO2•), and alkoxyl (RO•) radical, and which were included some
non-radical toxic species, such as H2O2, HOCl, fatty acid hydroperoxides
(FAOOH), reactive aldehydes, singlet oxygen (1O2) and other compounds
(Buonocore & Groenendaal, 2007; Jezek & Hlavata, 2005). RNS were radical
nitrogen-based molecules. The common primary RNS was nitric oxide (NO•)
and nitrogen dioxide (•NO2). Nitric oxide was a reactive radical that acted as an
important oxidative biological signaling molecule in a large variety of diverse
physiological processes, including neurotransmission, blood pressure regulation,
defense mechanisms, smooth muscle relaxation and immune regulation
(Bauerova & Bezek, 1999; Ghafourifar & Cadenas, 2005; Valko et al., 2006).
Nitric oxide itself was unreactive with most biological molecules. They were
7
becoming toxic when converted to secondary reactive nitrogen species
(non-radical toxic species) by nitrosylation reactions (Buonocore et al., 2007).
The non-radical toxic species included peroxynitrite (ONOO-), N2O3,
N-nitrosoamines, S-nitrosothiols, nitrosated fatty acids, and nitric oxide transition metal ions complex (ion-NO•), such as (Fe3+-NO−) and (Fe2+-NO•)
(Aruoma et al., 2006; Jezek et al., 2005; Valko et al., 2007; Valko et al., 2006).
Characteristic and features of ROS and RNS are show in Table 2.1.
2.2.3 Sources of ROS and RNS
ROS and RNS were generated during exogenous and endogenous pathway
(Fig. 2.4). Exogenous sources of ROS and RNS were generated from radiation
such as UV light, X-rays, and gamma rays, and nongenotoxic agent (quinones,
nitroaromatics, bipyrimidiulium herbicides, polyhalogenated alkanes, phenols,
aminophenols, and iron) and present as pollutants in the atmosphere (ozone and
singlet oxygen) (Rahman et al., 2006; Shchepinov, 2007; Valko et al., 2007).
Besides
endogenous
sources
of
ROS
and
RNS
that
include
mitochondria-catalyzed electron transport reactions, nitric oxide synthases,
cytochrome P450 metabolism, peroxisomes, inflammation, and metal-catalyzed
reactions (Fridovich, 1995; Hanukoglu et al., 1993; Salvador et al., 2001; Shen
et al., 1996).
2.2.4 Oxidative or nitrosative stress damage of free radical
Oxidative stress or nitrosative stress occurs when the generation of ROS or
RNS in a system exceeds the system’s ability to neutralize and eliminate them.
The imbalance can result from a lack of antioxidant capacity caused by
disturbance in production or by an over-abundance of ROS or RNS from an
8
Table 2.1 Characteristic and features of ROS and RNS.
(Aruoma et al., 2006)
9
Fig. 2.4 Exogenous and endogenous sources of ROS and RNS.
(Rahman et al., 2006)
10
environmental or behavioral stressor (Ercal et al., 2001; Finkel & Holbrook,
2000; Valavanidis et al., 2006).
2.2.4.1 Oxidative or nitrosative damage to biomolecules
If was not regulated properly, the excess ROS or RNS can damage a cell’s
lipids, protein or DNA, inhibiting normal function. Because of this, oxidative
stress or nitrosative stress has implicated in a growing list of human diseases as
well
as
aging
process
(Buonocore
et
al.,
2007;
Mishra
&
Delivoria-Papadopoulos, 1999). Potential markers of oxidative or nitrosative
damage in vivo was shown in Table 2.2.
2.2.4.1.1 Oxidative or nitrosative damage to deoxyribonucleic acid
DNA in cellular nuclei was a key cellular component that was particularly
susceptible to oxidative or nitrosative damage by free radical. The hydroxyl
radical was known to react with all components of the DNA molecule,
damaging both the sugar-phosphate backbone and nucleobases. The most
extensively studied DNA lesion was the formation of 8- Hydroxyguanine
(8-OH-G), which was used extensively as a biomarker for cellular oxidative
stress and genotoxicity in living organisms was 8-hydroxy-20-deoxyguanosine
(8-OHdG) or its oxidation product 8-oxo-20-deoxyguanosine (8-oxodG) (Fig.
2.5) (Valko et al., 2006). Other hydroxyl radical attacks could be directed
toward the sugar-phosphate backbone of DNA, causing different lesions,
including apurinic sites where the base has been removed, fragmentation of
deoxyribose with single-strand breaks, and oxidation of the sugar moiety (Cadet
et al., 1999; Kasai, 1997; Kirakosyan et al., 2003; Wiseman & Halliwell, 1996).
11
Table 2.2 Potential markers of oxidative or nitrosative damage in vivo.
(Wiseman& Halliwell , 1996)
12
Fig 2.5 Reaction of guanine with hydroxyl radical.
(Valko et al., 2006)
13
2.2.4.1.2 Lipid
Lipid oxidation was a typical chain reaction induced by oxygen in the
presence of initiators such as heat, free radicals, light, photosensitizing pigments
and metal ions and generally through a three-phase process (initiation,
propagation and termination) (Fig. 2.6) (Laguerre et al., 2007; Rahman et al.,
2006; Valko et al., 2007). The major product of lipid peroxidation other than
malondialdehyde was 4-hydroxy-2-nonenal (HNE). Mondialdehyde (MDA) was
mutagenic in bacterial and mammalian cells and carcinogenic in rats.
Hydroxynonenal was weakly mutagenic but appeared to be the major toxic
product of lipid peroxidation (Dix & Aikens, 1993; Esterbauer et al., 1990).
Various pathways of lipid peroxidation are shown in Fig. 2.7.
2.2.4.1.3 Protein
Protein oxidation reactions involve various propagating radicals and the
results are oxidative modifications of amino acid side chains, reactive
oxygen-species-mediated peptide cleavage, and formation of carbonyl
derivatives of proteins. Most of protein oxidation reactions were irreversible.
When the oxidation of cysteine or methionine residues may lead to the
reversible formation of mixed disulphides between protein thiol groups (-SH)
and low molecular weight thiols, especiall of GSH (S-glutathiolation). Oxidative
or nitrosative modifications of protein amino acids are shown in Fig. 2.8 (Berlett
& Stadtman, 1997; Dean et al., 1997; Stadtman, 2004; Valko et al., 2006). The
accumulation of oxidized proteins in vivo may be due to an increase in the
steady state level of ROS/RNS and a decrease in the antioxidant capacity of an
organisms and a decrease in the ability to degrade oxidized proteins due to either
a decrease in the protease concentrations or to an increase in the levels of
14
Fig. 2.6 The overall process of lipid peroxidation.
(Ramalho & Jorge, 2006)
15
Fig. 2.7 Various pathways of lipid peroxidation.
16
Fig. 2.8 Oxidative or nitrosative modifications of protein amino acids.
(Mello & Kubota, 2007)
17
protease inhibitors (Dalle-Donne et al., 2005; Mello & Kubota, 2007; Valko et
al., 2006).
2.2.4.2. Oxidative or nitrosative stress and disease
Oxidative or nitrosative stress has implicated in various pathological
conditions involving cardiovascular disease, cancer, neurological disorders,
diabetes, ischemia or reperfusion and ageing. These diseases consist with two
groups, the one involves diseases characterized by pro-oxidants shifting the thiol
or disulphide redox state and impairing glucose tolerance also known
“mitochondrial
oxidative
stress”
conditions,
another
involves
disease
characterized by “inflammatory oxidative conditions” and enhanced activity of
either NAD(P)H oxidase leading to atherosclerosis and chronic inflammation or
xanthine oxidase-induced formation of ROS or RNS implicated in ischemia and
reperfusion injury (Dalle-Donne et al., 2005; Valko et al., 2006). In research
studies, biomarkers can be employed in reflecting environmental prooxidant
exposures and dietary antioxidant intake or serving as a surrogate measure of a
disease process. Evidence for the association of oxidative or nitrosative stress
with acute and chronic diseases lies in validated biomarkers of oxidative stress.
Such biomarkers have to be objectively measured and evaluated on healthy and
ill subjects for long periods (Dalle-Donne et al., 2003; Dalle-Donne et al., 2005;
Dalle-Donne et al., 2006). Human diseases that were found to be associated with
increased oxidative stress on the basis of potential biomarkers of oxidative
damage shown in Table 2.3.
2.2.4.2.1 Diabetes
Diabetes was a chronic disease, which occurs when the pancreas did not
18
Table 2.3 Biomarkers of oxidative/ nitrosative damage associated with some human
disease.
(Dalle-Donne et al., 2006)
19
produce enough insulin, or when the organism couldn’t use the insulin. Diabetes
mellitus could be divided into two types: type 1 diabetes (previously known as
insulin-dependent or childhood-onset diabetes) was characterized by a lack of
insulin production and type 2 diabetes (formerly called non-insulin-dependent or
adult-onset diabetes) was caused by the organism’s ineffective use of insulin.
Diabetes lead to chronic extracellular hyperglycemia resulting in tissue damage
and pathophysiological complications, involving heart disease, atherosclerosis,
cataract formation, peripheral nerve damage, retinopathy and others (Aruoma et
al., 2006; Ceriello et al., 1993; Januszewski et al., 2003; Johansen et al., 2005).
Hyperglycemia in an organism stimulates ROS or RNS formation from a variety
of sources is shown in Fig. 2.9.
2.2.4.2.2 Neurodegenerative disorders
Oxidative stress has been implicated in various neurodegenerative
disorders and may be a common mechanism underlying various forms of cell
death including necrosis, apoptosis and excitotoxicity. The oxidative
stress-mediated neuronal loss may be initiated by a decline in the antioxidant
molecule glutathione. Glutathione plays multiple roles in the nervous system
including free radical scavenger, redox modulator of ion tropic receptor activity,
and possible neurotransmitter. The cell death in distinct neuronal populations,
can initiate as its depletion can enhance oxidative stress and may also increase
the levels of excitotoxic molecules (Berry & Hare, 2004; Dhalla et al., 2000;
Valko et al., 2007). Potential causal factors leading to oxidative stress in various
neurodegenerative disorders and mechanisms are shown Table 2.4 and Fig. 2.10,
respective.
20
Fig. 2.9 Hyperglycemia in an organism stimulates ROS or RNS formation from a
variety of sources.
(Johansen et al., 2005)
21
Table 2.4 Potential causal factors leading to oxidative stress in various
neurodegenerative disorders.
(Bains & Shaw, 1997)
22
Fig. 2.10 Mechanism of oxidative stress in neurodegenerative disorders.
(Bains & Shaw, 1997)
23
2.2.4.2.3 Cardiovascular disease
The free radical-induced oxidative stress in cardiac and vascular myocytes
has been linked with cardiovascular tissue injury. And free radical-induced
oxidative stress caused various cardiovascular diseases such as atherosclerosis,
ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy
and congestive heart failure. The sources of free radical in cardiovascular
system include (A) uncoupling of mitochondrial oxidative phosphorylation. (B)
The xanthine-oxidoreductase (XOR) system, include two enzymatic forms XD
(xanthine dehydrogenase) and XO (xanthine oxidase). XD was the form that
predominates in purine catabolism resulting in the synthesis of the antioxidant
uric acid. The XO form is associated with the synthesis of large amount of ROS
and RNS, which at low levels are important second messengers but at high
levels have microbicidal action. (C) Uncoupling of NO• synthesis. The
endothelial nitric oxide synthase (eNOS) with the deficiency of cofactors
L-arginine and BH4 ((6R)-5,6,7,8-tetrahydrobiopterin) switches from a coupled
state to an uncoupled oxide. Superoxide can further react with pre-formed NO•
and generate oxidizing agent peroxynitrite. (D) Activation of NAD(P)H oxidase
system by various mediators. The enzyme complex was activated in response to
a variety of vasoactive (Angiotensin I, Ang II), inflammatory (IL-6, TNF-α),
and growth factors (Berry et al., 2004; Huang et al., 2005; Molavi & Mehta,
2004; Valko et al., 2007). The pathways of ROS and RNS generation in
cardiovascular system are shown in Fig. 2.11.
2.2.4.2.4 Cancer
ROS or RNS induces a cellular redox imbalance which has been found to
24
Fig. 2.11 The pathways of ROS/RNS generation in cardiovascular system.
25
be present in various cancer cells are compared with normal cells. When
produced in excess, they play a role in the cause of cancer. ROS or RNS
mediated lipid peroxides are of critical importance because they participate in
chain reactions that increase damage to bio-molecules including DNA. DNA
attack gives rise to mutations that may involve tumor suppressor genes or
proto-oncogenes, and this is an oncogenic mechanism. In vivo, carcinogenesis
was a complex multi-sequence process leading a cell from a healthy to a
precancerous state and finally to an early stage of cancer. Theories of
carcinogenesis described cancer as a “disease of cell differentiation”, “stem cell
disease” or “single cell origin”. Commonly, two key mechanisms and three
stages model have been proposed for the induction of cancer. The first
mechanism for the induction of cancer was increased DNA synthesis and
mitosis by non-genotoxic carcinogens may induce mutations in dividing cells
through misrepair. The second mechanism was imbalance of cell proliferation
and cell death. If the damage to DNA was too great, there exists an important
process that eliminates altered cells selectively and this process was called
apoptosis. The three stages for the induction of cancer can be described by
initiation, promotion and progression. ROS /RNS can act in all these stages of
carcinogenesis show in Fig 2.12. (Trueba et al., 2004; Valko et al., 2007; Valko
et al., 2006)
2.3 Antioxidant
2.3.1 Antioxidant mechanisms
Antioxidants were divided into two groups depending on their mechanism
of action. The first was to protect which target lipids from oxidation initiators
(also known as preventive antioxidants hinder ROS formation or scavenge
26
Fig. 2.12 Three stages model of carcinogenesis.
27
species responsible for oxidation initiation) or by stalling the propagation phase
(also known as ‘chain breaking’ antioxidants which intercept radical oxidation
propagators
or indirectly participate in stopping radical chain propagation)
(Table 2.5) (Cadenas, 1997; Somogyi et al., 2007; Valko et al., 2007).
2.3.1.1 Preventive antioxidants
Preventive antioxidants reduce the rate of chain initiation, and chain
breaking antioxidants interfere with chain propagation. There were many
different pathways for preventive antioxidants through the removal of available
oxidation initiators. These major pathways include chelation of transition metals,
singlet oxygen deactivation, enzymatic free radical detoxification, UV filtration,
antioxidant enzyme, etc (Somogyi et al., 2007; Valko et al., 2007).
2.3.1.1.1 Transient metal chelators
The mechanism of transient metal chelators was maimly though the
inhibition of DNA strands breakage. Chelators of transition metals such as iron
can prevent oxidation by forming complexes or coordination compounds with
the metals. These chelators might be proteins including transferrin, ferritin,
lactalbumin, ceruloplasmin and albumin. Some compounds such as EDTA,
phenolic acids and flavonoids were also known for their transition metal
chelation capacity (Laguerre et al., 2007; Somogyi et al., 2007).
2.3.1.1.2 Singlet oxygen quenchers
Quencher of singlet oxygen has two types of quenching, one for chemical
quenching and physical quenching. Chemical quenching was a term used to
signify that an actual chemical reaction has occurred. Hydroperoxide formation
28
Table 2.5 Preventive antioxidants and chain breaking antioxidants.
(Somogyi et al., 2007)
29
was a kind of chemical quenching. The reactions can be described by the
following equation:
1
O2 + LH → LOOH
Physical quenching is the removal of the excitation energy from singlet
oxygen without any chemical changes. Carotenoids, especially β-carotene are
the most efficient molecules for singlet oxygen quenching. The mechanism of
action occurs through deactivation of 1O2 into 3O2. Then β-carotene is excited by
the extra energy into its excited state (β-carotene*), and then relaxes into its
ground state (β-carotene) by losing the extra energy as heat (Laguerre et al.,
2007; Valko et al., 2007).The reactions are described by the following equation:
1
O2 + β-carotene→ 3O2 + β-carotene*
β-carotene* →β-carotene + heat
2.3.2.1 Chain breaking antioxidants
In general, chain breaking antioxidants act by reacting with peroxyl
radicals, and they have two groups. The first was donor antioxidant. This
primarily includes mono- or poly-hydroxylated phenol compounds with
different substituents on one or several aromatic rings. And donor antioxidants
have two types. One is hydrophilic chain breakers such as fruits, vegetables, and
tea, coffee, and antioxidant reactions occurred in cytosolic, mitochondrial and
nuclear compartments. The other is hydrophobic chain breaking antioxidants
such as tocopherols (vitamin E) and the reactions occurred in cell membranes
where they inhibit or interrupt chain reactions of lipid peroxidation. The second
group of chain breaking antioxidants is sacrificial antioxidant such as nitric
oxide (Laguerre et al., 2007; Somogyi et al., 2007; Valko et al., 2007).
30
Antioxidant fates of nitric oxide on low density lipoprotein oxidation are shown
in Fig. 2.13.
2.3.2 Antioxidant defense in vivo
The effect of free radical is balanced by the antioxidant action of
antioxidant enzymes, as well as by non-enzymatic antioxidants. The term
antioxidant can be serve as a lable for any substance whose presence, even at
low concentrations, delays or inhibits the oxidation of a substrate. A good
antioxidant can specifically quench free radicals, chelate redox metals, and
interact with other antioxidants within the “antioxidant network” (Valko et al.,
2007). A relevant relationship between free radical and antioxidant was shown
in Fig. 2.14.
2.3.3.1 Antioxidant enzymes
Natural antioxidant enzymes produced in the body provide an important
defense against free radicals. They include superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), heme oxygenase and some low
molecule-weight protein system such as thioredoxin system, peroxiredoxins ,
and glutaredoxins are shown in Fig. 2.15 (Laguerr et al., 2007; Valko et al.,
2007).
2.3.3 Non-enzyme antioxidant
Non-enzymatic antioxidants involve water-soluble antioxidants such as
Vitamin C or phenolic compounds, and lipid-soluble antioxidants such as
Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic
acid) (Valko et al., 2006).
31
Fig. 2.13
Antioxidant fates of nitric oxide on low density lipoprotein oxidation.
(Rubbo et al., 2000)
32
Fig. 2.14
Relevant relationships between free radical and antioxidant.
(Moller et al., 2007)
33
(A)
(B)
Fig. 2.15 Antioxidants enzymic (A) Intracellular and extracellular distribution, (B)
Enzymatic degradation of reactive oxygen species.
(Rahman et al., 2006)
34
2.3.3.1 Vitamin C
Vitamin C, also known as ascorbic acid, was considered as an indicator of
the quality of food processing, due to its high degree of water solubility and low
stability during heat treatment. All plants and animals, except humans, can
synthesize ascorbate de novo; animals also can obtain vitamin C through their
diet. Ascorbate functions as a reductant for many ROS, such as preventing lipid
hydroperoxide formation in plasma lipoproteins, e.g., LDL, by reducing
α-tocopherol radicals formed upon reaction with lipid peroxyl radicals.
Ascorbate also protects lipids in cell membranes by this mechanism.
Intracellularly, in the aqueous phase, ascorbate and GSH act in concert to
protect the cell from oxidative damage (Fig. 2.16.) (Blokhina et al. 2003; Carr et
al., 2000; Kojo, 2004; Valko et al., 2004; Valko et al., 2006).
2.3.3.2 Vitamin E
Vitamin E is the collective name for a set of eight related tocopherols and
tocotrienols. Of these, α-tocopherol has been most studied as it has the highest
bioavailability, with the body preferentally absorbing and using this form. It has
been claimed that α-tocopherol is the most important lipid-soluble antioxidant,
and that it protects cell membranes from oxidation by reacting with lipid
radicals produced in the lipid peroxidation chain reaction. This would remove
the free radical intermediates and prevent the oxidation reaction from continuing.
The oxidized α-tocopheroxyl radicals produced in this process may be recycled
back to the active reduced form through reduction by other antioxidants (Carr et
al., 2000; Cerqueira et al., 2007)(Fig. 2.17).
35
(A)
(B)
Fig. 2.16
Ascorbic acid functions.(A) Various forms of ascorbic acid and its
reaction with radicals (R•). (B) Ascorbate-glutathione antioxidant
systems.
(Blokhina et al. 2003;Valko et al., 2004 )
36
Fig. 2.17 (A) Chemical structure of Vitamin E. (B) Antioxidation mechanism of
α-tocoferol.
(Cerqueira et al., 2007)
37
2.3.3.3 Carotenoids
Carotenoids (Car) are pigments that are found in plants and
microorganisms. All carotenoids posses a polyisoprenoid structure, a long
conjugated chain of double bond and a near bilateral symmetry. Structures of
some common carotenoids are shown in Fig 2.19 (Rao & Rao, 2007). Due to the
presence of the conjugated double bond, carotenoids can undergo isomerization
to cis-trans isomers. The antioxidant activity of carotenoids primarily attributes
to the conjugated double-bonded structure. Three mechanisms were proposed
for the reactions of free radicals (ROO•, R•) with carotenoids: (i) radical
addition, (ii) hydrogen abstraction from the carotenoid and (iii) electron-transfer
reaction (Fig. 2.18) (Valko et al., 2004)
2.3.3.4 Thiol antioxidants — glutathione and lipoic acid
Glutathione is the major thiol antioxidant and a tripeptide. It is considered to be
the major thiol-disulphide redox buffer of the cell. The reduced form of
glutathione is GSH, glutathione, and the oxidised form is GSSG, glutathione
disulphide, are shown in Fig. 2.19 (Valko et al., 2006). The oxidative or
nitrosative stress leads to rapid modification of protein sulphydryls (protein-SH).
The two-electron oxidation yields sulphenic acids (protein-SOH) and
the-electron oxidation yields thiyl radicals (protein-S•). Then the sulphenic acid
and thiyl radicals react with GSH to form S-glutathiolated protein by the
glutathione cycle. If the process of oxidation of protein sulphydryls is not
trapped by GSH, further oxidation leads to the formation of irreversibly
oxidized forms such as sulphinic (protein-SO2H) and sulphonic (protein-SO3H)
acids are shown in Fig. 2.20 (Valko et al., 2006).
38
Fig. 2.18
Structure of carotenoids.
(Cerqueira et al., 2007)
39
Fig. 2.19 Structures of of reduced (GSH) and oxidizes (GSSG) glutathinone.
40
Generally, the antioxidant capacity of thiol compounds is due to the
sulphur atom which can easily accommodate the loss of a single electron. The
main protective roles of glutathione inxlude acting as a cofactor of several
detoxifying enzymes which against oxidative stress, participating in amino
acid transport through the plasma membrane, directly scavenging hydroxyl
radical and singlet oxygen, detoxifying hydrogen peroxide and lipid peroxides
by glutathionperoxidase, and regenerating the most important antioxidants,
vitamins C and E back to their active forms (Nordberg & Arner, 2001; Valko et
al., 2006). The various pathways of glutathione (GSH) and other antioxidants
are shown in Fig. 2.21.
α-Lipoic acid (ALA); a disulphide derivative of octanoic acid is both
water and fat-soluble. Therefore, ALA is widely distributed in both cellular
membranes and the cytosol. It is readily absorbed from the diet and converted
rapidly in many tissues to its reduced dithiol form, dihydrolipoic acid (DHLA)
(Fig. 2.22). It is an essential prosthetic group in the dihydrolipoyl transacetylase
(E2) component of α-keto acid dehydrogenase complexes in mitochondria and is
covalently bound to a specific lysine side chain to form a lipoamide-like moiety.
Dihydrolipoic acid (DHLA) reduced from free ALA has powerful reductive
capacity and their antioxidant functions include metal-chelation with ions such
as Cu (II) and Fe (II), quenching of reactive oxygen species, repair of oxidized
proteins and regeneration of other antioxidants, including GSH, ascorbate, and
vitamin E (Nordberg & Arner, 2001; Smith et al., 2004; Valko et al., 2006).
2.3.3.5 Phnolic compounds
Polyphenolic compounds constitute one of the most commonly occuring
41
Fig. 2.20 Role of GSH in oxidation of protein sulphydry groups.
42
Fig. 2.21 Relationship of GSH and other antioxidants.
43
Fig. 2.22 Structure of lipoic acid and dihydrolipoic acid
44
and ubiquitous groups of plant metabolites and represent an integral part of
human diet. The most commonly occurring ones in foods are flavonoids, tannins
and phenolic acids. The multiple properties of these phytochemicals have made
them more attractive, as they can modulate various aspects of disease like lipid
peroxidation
involved
in
atherogenesis,
thrombosis,
carcinogenesis,
hepatotoxicity and a variety of disease conditions (Cai et al., 2004; Cai et al.,
2006).
Flavonoids present a great variety of structural specificities (Fig. 2.23) and
their basic structure contains a flavon nucleus (2-phenyl-benzo-γ-pyran)
consisting of benzene rings A and B combined by an oxygen-containing pyran
ring C. The differences in substitution on ring C give rise to the different classes
of flavonoids. Polyphenolic compounds can exercise their antioxidant activity in
several ways (Fig. 2.24). The first one is to act as “radical-scavengers”, such as
hydroxyl radical, singlet oxygen, dioxygen, and superoxide quenchers. The
second one is to act as terminators of free radical chains. Both these activities
were attributed to their hydrogen-donating ability. The final one is as metal ion
chelators and the activity of these phytochemicals is due to their binding metals
at two groups of their molecules; one is the ortho-dihydroxy (3’, 4’-diOH)
grouping in ring B and the other is the ketal structure 4-keto, 3-hydroxy or
4-keto and 5-hydroxyl in ring C. Their antioxidant activity was attributed to the
phenolic hydroxyls, particularly in the ortho-dihydroxy groups of the ring B or
A and the 2, 3-double bond in the ring C. Antioxidant activity increases with the
number of -OH groups or -OCH3 groups in rings A and B. On the contrary,
antioxidant activity decreases with glycosylation (Cai et al., 2004; Cai et al.,
2006; Laguerre et al., 2007; Tiwari, 2001).
45
Fig. 2.23 Skeletal structure of flavonoid and related structures.
( Tiwari, 2001)
46
(A)
(B)
Fig. 2.24
Flavonoids can exercise their antioxidant activity in several ways.(A)
Activity was attributed to their hydrogen-donating ability. (B) Metallic ion
complexation by flavonoids via the 30-40-o-diphenolic group in the B ring
(a) and ketol structures 4-keto, 3-hydroxy in the C ring (b) or 4-keto,
5-hydroxy in the C and A rings (c).
(Laguerre et al., 2007; Tiwari, 2001)
47
Natural tannins are high molecular weight and have many phenolic groups.
Tannins are commonly divided into condensed tannins, hydrolyzable tannins
and phlorotannins, as illustrated by the representative compounds shown in Fig.
2.25. Condensed tannins are mainly the oligomers and polymers of catechin
derivatives, also known as proanthocyanidins. Hydrolyzable tannins possessed a
central core of polyhydric alcohol such as glucose. The hydroxyl groups of the
carbohydrate are partially or totally esterified with phenolic groups such as
gallic acid (in gallotannins) or ellagic acid. Phlorotannins were oligomers of
phloroglucinol are found only in marine brown algae. Condensed tannins and
hydrolyzable tannins are powerful antioxidant agents because they possess a
great number of hydroxyl groups, especially many ortho-dihydroxy or galloyl
groups (Bouchet et al., 1998; Cai et al, 2006; Yokozawa et al., 1998).
48
Fig. 2.25
Typical tannins (polyphenolics). 1 is a typical procyanidin or condensed tannin (PC), made up of catechin and epicatechin. 2 is a
hydrolyzable tannin (polygalloyl glucose), made up of a glucose core esterified with gallic acid residues. 3 is a phlorotannin
(tetrafuhalol A), made up of phloroglucinol subunits.
(Hagerman et al., 1998)
49
CHAPTER 3 MATERIALS AND METHODS
3.1 Experimental procedures
Tested herbal medicines
Ground, extracted
Boiling water
Methanol extract of herb was dissolved
in deionized water
Organic solvents
Methanol
Ethyl acetate
Determination of antioxidant activities
Sequential extraction with
n-hexane, diethyl ether, ethyl
acetate and 1-butanol
Contents of antioxidant components
1. Radical-scavenging activity
1 Contents of total phenolic
1.1 DPPH radical scavenging activity
2 Contents of flavonoids
1.2 ABTS cation radical scavenging activity
3 Content of condensed tannins
1.3 Superoxide anion radical scavenging activity
2. Ferric reducing antioxidant power (FRAP)
3. Ferrous ion-chelating capacity
4. Inhibition of lipid peroxidation assay
Fig. 3.1 Experimental procedures.
50
3.2 Tested herbal medicines
According to the Cheng (2004) “screening of anti-tyrosinase, antioxidant
and anti-acne activities of thirty-one herbal medicines” which found high
phenolic contents herbal were B. javanica and P. chinens. These herbal
medicines were gotten from “Fu-gi” herbal medicine store (No.246, Sichang St.,
Wanhua District, Taipei City 108, Taiwan). Two tested herbal medicines were
shown in Table 3.1.
3.3 Chemicals
2,2’-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), butylated
hydroxyanisole (BHA), 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
Acid (trolox), phenazine methosulfate (PMS), β-nicotinamide adenine
dinucleotide (NADH), nitro blue tetrazolium (NBT), Hydrogen peroxide (H2O2),
ferrozine, gallic acid, vanillin, quecetin and catechin were purchased from
Sigma Chemical Co. (St. Louis, MO, USA).
1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium persulfate (K2S2O8),
sodium chloride (NaCl), potassium dihydrogen phosphate KH2PO4), sodium
dihydrogen phosphate (Na2HPO4), potassium chloride (KCl ), sodium hydroxide
(NaOH), hydrogen chloride (HCl), 2, 4, 6-tripyridyl-s- triazine (TPTZ), sodium
acetate trihydrate, glacial acetic acid, 2-Thiobarbituric Acid (TBA), sodium
carbonate
(Na2CO3),
aluminium
Chloride(AlCl3),
lecithin,
ferrous
chloride(FeCl2) and Folin-ciocalteu’s reagent were purchased from WAKO
Chemical Co. (Osaka, Japan).
Ascorbic acid was obtained from Merck (Darmstadt, Germany). Ferric
chloride 6-hydrate (FeCl3．6H2O) and Trichloroacetic acid (TCA) were obtained
51
from J. T. Baker (Phillipsburg, U.S.A.). Tris (hydroxymethyl) aminomethane
(Tris) was obtained from BioShop (Burlington, Canada). Methanol, 1-butanol,
n-hexane, diethyl ether, and ethyl acetate were obtained from Echo Chemical Co
Ltd. (Taiwan, R.O.C).
3.4 Preparation of extracts from herb
3.4.1 Boiling water extraction of herb
The samples were prepared according to Saha et al. (2004) with
modifications. Tested herbal medicines were dried in the dark at 40℃ (Oven,
Hotech Shaker bath, Model 902, Taipei, Taiwan) for a week. 30g of herb were
ground to powder (18 mesh) with a mechanical grinder (Mill and sieving,
YU-CHI, Taiwan) and extracted with 600 ml boiling water for 30 min (Water
bath, HOTECH 903, Taipei, Taiwan). Then the extract was cooled to room
temperature and centrifuged at 10000 g for 20 min (HitachiCF15D2, Tokyo,
Japan). The suspension was filtered through Whatman No.2 filter paper to
clarify. The residue was then extracted with two additional 600 ml of boiling
water as described above. The combined extracts were then rotary evaporated
(Rotavapor, Büchioll, Flawil, Switzerland) and then lyophilized (Freezer Dryer,
Virtis, UNITOP 600SL, Gardiner, Montana, U.S.A.). The lyophilized powder
was stored in a moisture-proof chest.
3.4.2 Organic solvents extraction
The samples were prepared according to Tseng et al. (2006) with
modifications. 30 g samples of herbal medicines powder were shaking at 120
rpm for 24 h at room temperature with 600 mL either of methanol or ethyl
acetate, respectively. Then the extract was centrifuged at 10000 g for 20 min.
52
The suspension was filtered through Whatman No.2 filter paper to clarify. The
residue was extracted with two additional 600 ml of either methanol or ethyl
acetate, respectively, as described above. The combined extracts were then
rotary evaporated at 40°C to dryness. The dry powder was stored in a
moisture-proof chest.
3.5 Determination of antioxidant activities
3.5.1 Radical-scavenging activity
3.5.1.1 DPPH radical scavenging activity
Antioxidant properties especially radical scavenging activities was very
important, because of the free radicals have deleterious effects in foods and
biological systems (Gulcin et al., 2005). DPPH is one of a few stable,
commercially available organic nitrogen radicals. Because of its odd electron,
DPPH gives a strong absorption maximum at 517 nm (purple color). When the
hydrogen donor or free radical-scavenging antioxidant was present, that leads to
the odd electron of the radical becomes paired off, the absorbance at 517 nm
was decreased, and the resultant
decolorization was stoichiometric with
respect to the number of electrons captured (Gulcin et al., 2005; Yamaguchi et
al.,1998). The structure of DPPH· and its reduction by an antioxidant is shown
in Fig. 3.2.
DPPH· scavenging activity was measured in 96-well microplate according
to Shimada et al. (1992) with modifications. 50 μl of sample solutions and 150
μl of 400 μM DPPH methanolic solution were added into each well. The 96-well
microplate was incubated at 25℃ for 90 min and then measured the absorbance
at 517 nm (UV-spectrophotometer, UNICAM, HeλIOSα, Cambridge,
53
Fig. 3.2
Scheme for scavenging the DPPH radical by an antioxidant.
RH, antioxidant.
（Yamaguchi et al.,1998）
54
Massachusetts, U.S.A.). BHA and ascorbic acid was used as a positive control.
Scavenging activity (%) was obtained by the following equation:
Scavenging activity (%) = [Acontrol－( Atest－A blank )] / Acontrol × 100
Where Acontrol is the absence of sample solutions, Atest is the absorbance of
the test sample and Ablank is the absence of DPPH methanolic solution.
EC50 of the extract was the concentration of the test sample at which 50%
scavenging activity was obtained. The values were calculated from the
concentration-effect linear regression curve. Ascorbic acid and trolox were used
as standards and the activities were also expressed as trolox equivalents (TE) /
ascorbic acid equivalent (AE) in micromole per gram of extracts/ subfractions.
3.5.1.2 ABTS cation radical scavenging activity
ABTS·+, the oxidant, was generated by persulfate oxidation of ABTS
(Huang et al., 2005). It has absorption maxima at wavelengths 645 nm, 734 nm
and 815 nm, as reported previously. In the presence of the stable radical ABTS·+
is reduced to be colorless colored. The absorption strength was decreased
stoichiometric ally with respect to the number of electrons captured (Re et al.,
1999). The structure of ABTS and its reduction by an antioxidant is shown in
Fig. 3.3. ABTS·+ was generated by reacting ABTS (2 mM) solution with K2S2O8
(19.6 mM, final concentration) in the dark for 15–16 h before use (Loziene et al.,
2007). For the assessment of samples, the ABTS·+ solution was diluted with
phosphate buffer to obtain the absorbance of 0.800 ± 0.030 at 734 nm. After 10
μl extract solution were added to 1.0 ml ABTS·+, the absorbance at 734 nm was
recorded after 10 min. BHA and ascorbic acid was used as a positive control.
The percentage decrease of the absorbance at 734 nm was calculated by
55
Fig. 3.3 Scheme for scavenging the ABTS·+ by an antioxidant.
(Childs & Bardsley, 1975)
56
equation:
Scavenging activity (%) =
[Acontrol－( Atest－A blank )] / Acontrol × 100
the test sample and Ablank is the absence of ABTS·+ solution.
3.5.1.3 Superoxide anion radical scavenging activity
Superoxide anion, a reduced form of molecular oxygen, has been
implicated in the initiating oxidation reactions associated with aging. A
superoxide radical was generated in a PMS-NADH system by oxidation of
NADH and the reduction of NBT. The reaction of NBT with superoxide radical,
results in the formation of a highly colored diformazan (Fig. 3.4).
This reaction was conveniently monitored spectrophotometrically by an
increase in absorbance at 560 nm (Liu & Ng, 2000; Qi et al.,2006).
Measurement of superoxide anion scavenging activity of samples was modified
form the method described by Liu and Ng (2000). The superoxide radicals were
generated form a mixture containing 150 μl of 200 μM NBT, 624 μM β-NADH,
80 μM PMS and samples in 16 mM Tris-HCl buffer (pH 8.0). The reaction was
initiated by adding PMS and incubated at 25℃ for 10 min. The absorbance was
then determined at 560 nm. Scavenging activity (%) was calculated by the
57
Fig. 3.4 Scheme for superoxide anion was generated in a PMS- NADH system.
58
following equation:
Scavenging activity (%) =
[Acontrol－( Atest－A blank )] / Acontrol × 100
test sample and Ablank is the absence of PMS solution.
3.5.2 Ferric reducing antioxidant power (FRAP)
Ferric ion reducing antioxidant power (FRAP) assay which was measures
the combined antioxidant effect of the non-enzymatic defenses in biological
fluids was useful in providing an index of ability to resist oxidative damage. The
assay takes advantage of electron-transfer reactions. In this a ferric salt, Fe (III)
(TPTZ) 2Cl3, was used as an oxidant. The principles of FRAP was at low pH,
when a ferric-tripyridyltriazine complex was reduced to the ferrous form by
reductants (antioxidants) in the mixture, an intense blue color with an absorption
at 593 nm develops. In this assay, excess ferric ion was used, and the
rate-limiting factor of ferrous-tripyridyltriazine formation, and for this reason
color, was the reducing/antioxidant of the sample (Benzie & Strain, 1996;
Benzie & Strain, 1997; Huang et al., 2005). The structure of Fe (III) (TPTZ) 2Cl3
and its reduction by an antioxidant was shown in Fig. 3.5.
The FRAP assay of samples were determined by the method of Benzie and
59
Fig. 3.5 Scheme for Fe (III) (TPTZ) 2Cl3 and its reduction by an antioxidant.
(Huang et al., 2005)
60
Strain (1996) with modifications. The FRAP reagents included 300 mM acetate
buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl. FRAP reagent was prepared as
required by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml
(20mM) FeCl3．6H2O solution. The FRAP assay is performed using the FRAP
reagent preheated to 37 oC. To 30 μl sample or standard are added 900 μl FRAP
solution and 90 μl water. The mixture is incubated at 37oC for 4 min (HOTECH
903, Taipei, Taiwan). Absorbance at 593 nm is determined relative to a reagent
blank that is also incubated at 37℃. Antioxidant power of extracts/ fractions was
determined against a standard curve of ferrous sulphate or trolox.
3.5.3 Ferrous ion-chelating activity
It has been well recognized that transition metal ions such as those of iron
and copper were important catalysts for the generation of the first few free
radicals to initiate the radical chain reaction or the radical-mediated lipid
peroxidation. This method is based on the formation of a colored ferrozine-Fe2+
complex which has a strong absorbance at 562 nm (Zhao et al., 2008). In the
presence of chelating agents such as antioxidant compounds, the ferrozine-Fe2+
complex formation was disrupted, resulting in a decrease in the violet color of
the complex. The measurement of color reduction therefore allows an estimation
of the metal chelating activity (Gulcin et al., 2005).
The equation of ferrozine reduction by ferrous ion :
Fe2+ + ferrozine →ferrozine-Fe2+ complex ( violet )
The chelating activity for ferrous ions was measured following the
ferrozine method with minor modifications (Zhao et al., 2008). The reaction
61
mixture contained 100 μl of samples and 10 μl of 2 mM ferrous chloride. The
reaction was initiated by the addition of 5 mM ferrozine (20 μl), and the total
volume was adjusted to 500 μl with methanol solution. Then, the mixture was
shaken vigorously and incubated at room temperature for 10 min. The
absorbance of the solution was measured at 562 nm. The activities results were
expressed as micromole of EDTA equivalents (EDTAE) per gram of extracts /
fractions.
3.5.4 Inhibition of lipid peroxidation
In biological systems, iron salts are always bound to proteins, membranes,
nucleic acids, or low-molecular-weight chelating agents. They play a role in the
biological redox system by binding to a number of extracellular proteins, such as
transferrin and ceruloplasmin. This reaction is important in the prevention of
oxidative damages. Yen et al. (1999) developed an experimental system to
evaluate lipid peroxidation by oxygen free radicals in biological and medical
research. This method is based on the Fenton reaction and detects
non-enzymatic autoxidation of lipid, and the production of thiobarbituric acid
reactive substances (TBARS). The assay principle is as follows: the ascorbic
acid can reduce Fe3+ to Fe2+, H2O2 then interacts with iron (II), to form hydroxyl
radicals by the Fenton reaction. Hydroxyl free radicals attack unsaturated lipids
to form malonaldehyde (MDA), which is detected by its ability to react with
thiobarbituric acid (TBA) to form a pink chromogen (Fig. 3.6).
The lipid peroxidation assay was based on the method described by Chen et
al. (2007) with modification. Lecithin was sonicated in an ultrasonic cleaner in
10 mM phosphate buffer (pH 7.4) for 15 min at ice bath, and was mixed with
62
Fig. 3.6 The reaction of inhibition lipid peroxidation.
(Laguerre et al., 2007; Yen et al. 1999)
63
FeCl3, H2O2, ascorbic acid and samples to produce a final concentration of
lecithin 2.5 mg /ml, 125 μM FeCl3, 125 μM H2O2 and 125 μM ascorbic acid.
The mixture was incubated for 1 h at 37℃. The oxidation of lecithin was
measured by the TBA method.
The absorbance of the sample was read at 532 nm against a blank which
contained all reagents except lecithin. Inhibition of peroxidation (%) was
calculated by the following equation:
Inhibition of peroxidation ( % ) = [Acontrol－( Atest－A blank )] / Acontrol × 100
Where Acontrol was the absorbance of control (the absence of sample
solutions), Atest was the absorbance of tested sample, and Ablank was the
absorbance of blank (without lecithin).
3.6 Contents of antioxidant components
3.6.1 Contents of total phenolic
Plant foods rich in phenolic compounds have often been associated with
decreased risk of developing diseases, including cancer, heart diseases,
hypertension and stroke, etc. (Silva et al., 2002). Total phenolic content was
determined through sequences of reversible one- or two- electron transfer
mechanism. Phenolic compounds under basic conditions adjusted by a sodium
carbonate solution (pH~10) were deprotonated into phenolate anions, which
could be rechuced with molybdenum in the Folin-ciocalteu’s reagent to blue
species, possibly (PMoW11O40)4- (Huang et al., 2005). Total phenolics content of
samples were determined by the method of Taga et al. (1984). Aliquots of
samples 100 μl were mixed with 2 ml of 2 % Na2CO3 were incubated for 2 min
64
at room temperature. Then, 100 μl of 50 % Folin-ciocalteu’s reagent was added.
After 30 min of reaction at room temperature, the absorbance at 750 nm was
determined. The results were expressed as gallic acid equivalents (GAE) in
milligrams per gram of extracts/ fractions.
3.6.2 Contents of flavonoids
Flavonoids were polyphenolic compounds that exist ubiquitously in plant
tissues in relatively high concentrations as sugar conjugates, mostly in
O-glycosidic form (Grubesic et al., 2007). The flavonoid content as estimated by
the AlCl3 method (Lamaison et al., 1990). The methanolic extract solution 200
μl was added to 200 μl of 2% methanolic AlCl3．6H2O. The absorbance was
measured 10 min later at 430 nm. The results were expressed as quecetin
equivalents (QE) in milligrams per gram of extracts/ fractions.
3.6.3 Content of condensed tannins
Some medicinal plants contain complex mixtures of both hydrolyzable and
condensed tannins. Condensed tannins are mainly the oligomers and polymers
of flavan-3-ols (catechin derivatives), also known as proanthocyanidins.
Colorimetric procedures such as the vanillin hydrochloric acid (HCl) assay and
have been generally used for the determination of condensed tannins in foods.
(Nakamury et al., 2003).
The principle of the vanillin-HCl assay is as follows: vanillin is protonated
in an acid solution, giving a weak electrophilic carbocation that reacts with the
flavonoid ring at the 6 or 8 positions. This intermediate compound is dehydrated
to give a red colored compound (Nakamury et al., 2003). The condensed tannins
were assayed colorimetrically by the method of Broadhurst and Jones (1978). To
65
150 μl of methanolic solution of condensed tannins, 150 μl of 0.5% vanillin
reagent were added. 150 μl of 4% HCl in methanol was used as a blank. The
absorbances of samples and blank were read at 500 nm after standing for 20 min
at room temperature. Catechin was used as a standard in these experiments. The
results were expressed as catechin equivalents (CE) in milligrams per gram of
extracts/ fractions.
3.7 Preparation of extracts with organic solvents from crude methanol extracts
Preparation of extracts in organic solvents from crude extracts of methanol
by the method of Matsingou et al. (2003) with minor modifications. The crude
extracts of methanol was dissolved in water (40mg/ml) at room temperature and
reextracted sequentially with 200 mL of n-hexane, diethyl ether, ethyl acetate,
and 1-butanol, by shaking at 100 rpm for 5 min three times or more, until
complete decoloration of the organic phase. The aqueous layers from each
extraction were collected and combinded to be the “water fraction”. All the
fractions were collected and then rotary evaporated at 40°C to complete dryness.
All the extract fractions were dissolved in methanol and subjected to
determination of antioxidant activities and components contents of antioxidant.
3.8 Statistical analysis
All tests were conducted in triplicate. Data are reported as means ± SD.
Analysis of variance and significant differences among means were tested by
one-way ANOVA and Duncan’s multiple range tests using SAS software. The
Pearson correlation analysis was also performed by SPSS to determine the
correlations among means and the differences and similarities among in term of
antioxidant activities, respectively.
66
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Bischofia javanica
4.1.1 Extraction yields from different solvents
Different solvent systems have been frequently used for extraction. The
extraction yield is dependent on the solvent and method of extraction, due to
compound of different polarity. Several extraction techniques have been
reported for extraction of phenolic compounds from different plants materials
using solvents with different polarities, such as methanol, water, ethyl acetate,
actone and petroleum ether (Chavan et al., 2001; Cheung et al., 2003; Singh et
al., 2002; Zuo et al., 2002). Water, methanol and ethyl acetate were used as
extraction solvents for B. javanica in this work. As shown in Table 4.1, the
yields varied with different solvents. The highest yield was obtained with
methanol, followed by boiling water and ethyl acetate. Similar result was
reported by Cheung et al. (2003). The extraction yields of Lentinus edodes and
Volvariella volvacea were with methanol, water, ethyl acetate and petroleum
ether. Singh et al. (2002) found methanol to be the best solvent for extracting
Punica granatum peel and seed as compared with water and ethyl acetate.
4.1.2. Antioxidant activities of various extracts from B. javanica
4.1.2.1 Radical-scavenging activity
It is well-known that free radicals play an important role in oxidative cell
damage, and cause a variety of pathological diseases. Antioxidants are able to
contribute electrons themselves, thereby forming stable free radicals and
suppressing further oxidation of biomolecules (Dziezak, 1986; Halliwell, 1991;
Kroyer, 2004; Sherwin, 1978; Valko et al., 2007). The radical-scavenging
67
Table 4.1 Extraction yield of B. javanica from different solvents.
Extraction yield (%)1,2,3
Solvents
Boiling water
11.5 ± 2.8 b
Methanol
23.0 ± 2.2 a
1.0 ± 0.3 c
Ethyl acetate
1. Data are expressed as mean ± standard derivation from triplicate experiments.
2. Extraction yield is defined as weight percentage of extract (g) per gram of dry
weight of B. javanica.
3. Values within the column followed by different letter by Duncan’s method were
significantly different (p< 0.05).
68
activity could be assayed by several test systems. However, variation has been
reported between the test systems for the estimation of radical-scavenging activity.
Therefore, at least two and more test systems such as hydroxyl radical scavenging
(deoxyrribose assays), organic radical scavenging (DPPH·, ABTS·+), superoxide
radical (β-NADH-PMS-NBT assays) (Aruoma, 2003; Frankel & Meyer, 2000;
Kroyer, 2004; Perezjimenez & Saura-Calixto, 2006; Sanchez-Moreno, 2002). Of all
these methods organic radical scavenging (DPPH, ABTS), superoxide radical (O2·-,
β-NADH-PMS-NBT assays) are commonly used for plant extracts. (Dastmalchi,
2008; Frankel & Meyer, 2000; Piccolella et al., 2008; Zhao et al., 2008; Zhou and
Yu, 2004; Zielińsk et al.,2008)
All the extracts of B. javanica were evaluated for their radical scavenging
activity against DPPH·, ABTS·+ and O2·- (Fig. 4.1). As observed in Fig 4.1, there
were differences in the DPPH· scavenging activity among the extracts of B.
javanica and they were displayed in a concentration-dependent scavenging manner.
The highest activity was obtained in the methanol extract, followed by boiling water
and ethyl acetate extracts, and the scavenging effects were 84.0%, 61.6% and 3.5%,
respectively, at the concentration of 100 μg/ml. The scavenging effect of methanol
extract was similar to those of ascorbic acid (88.7%) and BHA (89.7%) at
concentration of 100 μg/ml.
ABTS·+ scavenging activity of B. javanica extracts showed the trend similar to
that of DPPH· scavenging activity with concentration-dependent fashion (Fig. 4.1).
Among B. javanica extracts, methanol extract possessed the highest scavenging
activity against ABTS·+, followed by boiling water extract, however, ethyl acetate
extract exerted a weak activity. With regarded to superoxide anion scavenging
69
DPPH scavenging activity (%)
100
80
60
.
40
20
0
0
200
400
600
800
1000
Concentration (μg/ml)
80
60
40
+.
ABTS scavenging activity (%)
100
20
0
0
1000
2000
3000
4000
Superoxide anion radicalss cavenging activity (%)
100
80
60
40
20
0
-20
0
200
400
600
800
1000
1200
Fig. 4.1 Radical scavenging activities of different extracts of B. javanica.●, Boiling
water； ○, Methanol； ▼, Ethyl acetate；△, Ascorbic acid；■, BHA.
70
activity and the highest scavenging activities was found in methanol extract,
followed by boiling water extract, while ethyl acetate exerted no scavenging activity
(Fig. 4.1).
The high scavenging activity might be attributed to betulinic acid isolated
from CHCl3 extract of B. javanica (Wada and Tanaka, 2005), which was reported to
exhibit high scavenging activity against DPPH· and superoxide anion radicals (Rana
et al., 2005). In addition, the high scavenging activity might be attributed to geraniin,
corilagin and punicalagin isolated from actone extract of B. javanica (Tanaka et al.
1995), which was reported to exhibit higher scavenging activity against DPPH· and
superoxide anion radicals than those of gallic acid, rutin, quercetin, and BHA
(Kulkarni et al., 2004; Tabata et al., 2008).
Scavenging activities against DPPH·, ABTS·+ and O2·- of different extracts
from B. javanica, in terms of EC50 (concentration for decreasing 50% of radicals),
AEAC (ascorbic acid equivalent antioxidant capacity) and TEAC (trolox equivalent
antioxidant capacity) were also determined and are presented in Table 4.2. Lower
EC50 or high AEAC and TEAC value represent high radical scavenging activity.
Among three extract, methanol extract showed the most effective scavenging
activity against DPPH·, ABTS·+ and O2·-. It’s EC50, AEAC and TEAC value for
DPPH· scavenging activity were 50.4 μg/ml, 5298.1 μmol AE/g and 5664.9 μmol
TE/g, respectively, similar to that of ascorbic acid with no significant difference (P
>0.05) under the same experimental conditions. The result suggested that the
quenching DPPH· activity of methanol extract of B. javanica was as effective as
ascorbic acid. However, scavenging activity of against ABTS·+ was significantly
lower than those of ascorbic acid and BHA. On the other hand, methanol and
boiling water extracts had significantly higher (P <0.05) superoxide anion
71
Table 4.2 Radical scavenging activity of various extracts from B. javanica extracts.1,2,3,4
DPPH
Specimens
EC50
(μg/ml)
Boiling water
Methanol
Ethyl acetate
Ascorbic acid
BHA
AEAC
O2·-
ABTS
TEAC
(μmol AE/g) (μmol TE/g)
EC50
AEAC
TEAC
EC50
AEAC
TEAC
(μg/ml)
(μmol AE/g)
(μmol TE/g)
(μg/ml)
(μmol AE/g)
(μmol TE/g)
70.3 ± 3.1 b
3869.1 ± 73.8 c
4173.6 ± 53.0c
537.8 ± 10.9b
2675.6 ±
18.1c
4277.4 ± 31.8c
317.4 ± 1 2.5 a
49147.9 ± 1619.5 b
20636.6 ±
50.4 ± 1.2c
5298.1 ± 57.2b
5664.9 ± 41.1b
372.9 ± 6.1c
3409.3 ± 143.8b
5570.7 ± 253.5b
216.3 ± 16.9 b
600319 ± 5826.1 a
25931.5 ± 2834.3 a
316.6 ± 3.0a
774.3 ± 31.6d
768.3 ± 23.7d
1725.0 ± 48.0a
1224.1 ±68.2d
ND5
49.0 ± 0.7 c
5368.0 ± 109.7b
5715.1 ± 78.8b
224.9 ± 4.5 d
5285.1 ± 243.4a
9120.7 ±423.2a
41.3 ± 0.7d
6155.6 ± 32.8a
7090.0 ± 62.2a
228.2 ± 2.0 d
5002.4 ± 157.4a
8979.2 ±99.9a
943.6 ±
38.7d
2677.4
±
ND5
11.6 a
787.8 b
4793.7 ±
338.9 c
1022.8 ±
164.9 c
6238.3 ±
398.3c
1725.6 ±
193.8c
ND5
ND5
1. Each value was expressed as mean ± S.D. (n=3). Values within the column followed by different superscript by Duncan’s method were
significantly different (p<0.05).
2. EC50 meant the concentration (μg/ml) of sample required to scavenge 50% of radical.
3. AEAC meant ascorbic acid equivalent antioxidant capacity.
4. TEAC meant trolox equivalent antioxidant capacity.
5. ND meant not determined.
72
scavenging activity than ascorbic acid with regard to the EC50 or AEAC and
TEAC value.
The scavenging activity against DPPH· of the methanol extract of B.
javanica was found to be much higher than those against ABTS·+ and
superoxide anion. Similar results were also reported in Prunus cerasus
(Piccolella et al., 2008) and Agaricus bisporus (Savoie et al., 2008). The
dissimilar relative scavenging activities against different radicals may be due to
different quench mechanisms with the radical-antioxidant reaction (Wang et al.,
1998; Mathew and Abraham, 2006; Yu et al. 2002). Stereoselectivity of the
radicals, solubility of the extract in different testing systems, the stoichiometry
of reactions between the antioxidant compounds in herbs extracts might also
contributed to the variation among scavenging activity of methanol extract of B.
javanica against different radicals (Wang et al., 1998; Mathew and Abraham,
2006; Yu et al. 2002; Zhou and Yu, 2004).
4.1.2.2 Ferric reducing antioxidant power
Reducing power was associated with antioxidant activity and served for a
significant indicator of its potential antioxidant activity due to donate electron
and reduce the oxidized intermediate during oxidative damage (Yen and Chen,
1995). The ferric reducing antioxidant power assay (FRAP) of antioxidants can
be estimated by the reaction ability of Fe3+ to Fe2+ (Guo et al., 2003; Oktay et al.,
2003).
As showed in Table 4.3, the reducing power of B. javanica extracts from
different extraction solvent expressed as μmol Fe2+ and trolox equivalent /g
73
Table 4.3 Reducing activity of B. javanica extracts using FRAP method.
Specimens
(μmol Fe2+ /g)
FRAP-value1,2
(μmol trolox / g)
Boiling water
2528.7 ± 82.1c
1712.3 ± 54.7c
Methanol
3988.2 ±191.5b
2685.2 ± 127.7b
Ethyl acetate
1095.4 ± 47.4d
756.7 ± 31.6d
Ascorbic acid
11222.1 ±190.0a
7507.8 ± 126.7a
BHA
11754.8 ±147.2a
7863.0 ± 98.1a
1. Data expressed as mean ± standard deviation (n=3). Values within the column
followed by different letter by Duncan’s method were significantly different (p<
0.05).
74
extract (TEACFRAP). Reducing activity of all extracts showed notable differences
and the FRAP-values ranged from 1095.4 to 3988.2 μmol Fe2+ /g of 756.7 to
2685.2 μmol trolox/g. The highest FRAP-value, 3988.2 μmol Fe2+ /g (2685.2
μmol trolox /g), was found in methanol extract, indicating that methanol extract
of B. javanica had the stronger reducing activity, followed by boiling water and
ethyl acetate ones. However, under the same experimental conditions, the
FRAP-value of methanol extract were much lower than those of ascorbic acid
(11222.1 μmol Fe2+ /g and 7507.8 μmol trolox /g) and BHA (11754.8 μmol Fe2+
/g and 7863.0 μmol trolox /g). The trend of reducing power for three B. javanica
extracts was similar to the result of scavenging activities against DPPH·, ABTS·+
and superoxide anion radical.
Interestingly, TEAC FRAP value of methanol extract 2685.2 μmol trolox /g
was much lower than those obtained for its corresponding TEAC
μmol trolox /g), TEAC
ABTS
DDPH
(5664.9
(5570.9 μmol trolox /g) and TEAC superoxide
anion radical (25931.5 μmol trolox /g). Boiling water and ethyl acetate extracts
also displayed similar results. The similar trend was also reported in evaluation
of antioxidative activity of Indian medicinal plants (Surveswaran et al., 2007)
and ethylene-treated kiwifruits (Park et al., 2008). This dissimilar relative
antioxidant activity of natural plant products may be due to different
mechanisms and the complexity of the reaction kinetics (Ozgen et al., 2006).
4.1.2.3 Ferrous ion-chelating activity
It has been reported that transition metal ions, especially iron and copper,
were important catalysts for generation of first radicals via Fenton type reaction
which initiated the chain reaction or mediated lipid peroxidation (Valko et al.,
75
2005). Chelating agents, such as some phenolic compounds, may stabilize
transitions metal ions in the organism and inhibit radical generations,
consequently rescuing radical damage. As shown in Table 4.4, all B. javanica
extracts exhibited metal chelating activities. The values of the metal chelating
activity ranged from 7.5 to 59.2 μmol EDTAE / g. Methanol extracts showed the
highest metal chelating activity, 59.2 μmol EDTAE / g, similar to those of
ascorbic acid and BHA with no difference (P>0.05) under the same
experimental condition. The result suggested that the ferrous ion-chelating
activity of methanol extract of B. javanica was as effective as ascorbic acid and
BHA. Additionally, ethyl acetate extract showed the lowest ferrous
ion-chelating activity.
4.1.2.4 Inhibition of lipid peroxidation
Lipid peroxidation of cell membranes can increase lipid-phase surface
charge and formation of protein oligomers, and those enhance the in oxidative
stress of biological system. Malonaldhyde, one of the products of lipid
peroxidation, can be as a marker of oxidative stress and dctetected by TBA
method (Girotti, 1998; Siddhuraju and Becker, 2007). Inhibition of lipid
peroxidation by B. javanica extracts are presented in Fig. 4.2. Boiling water and
methanol extracts showed inhibition of lipid peroxidation at a concentration of
1000 μg/ml. On the contrary, the poorest inhibition was found in ethyl acetate.
IC50 values, were estimated by a linear regression algorithm, of these sample
followed the order: BHA > methanol extract > water extract and the IC50 were
87.3, 354.0 and 187.1 μg/ml, respectively (Table 4.5). Inhibition of lipid
peroxidation of antioxidants might be resulted from cheating metal ions, and
scavenging the hydroxyl radicals (Gutteridge, 1985). In this study, we incubated
76
Table 4.4 Ferrous ion-chelating capacity of various extracts from B. javanica.
Specimens
Metal chelating activities 1,2
(μmol EDTAE / g)
Boiling water
23.2 ± 1.7 b
Methanol
59.2 ± 3.7 a
Ethyl acetate
7.5 ± 2.4 c
Ascorbic acid
56.6 ± 3.4 a
BHA
51.5 ± 4.4 a
0.05).
2. EDTAE standard for EDTA equivalents.
77
Inhibition of lipid peroxidation activity
100
80
60
40
20
0
0
200
400
600
800
Fig. 4.2 Inhibition of lipid peroxidation of boiling water and methanol extracts
from B. javanica and BHA. Values are means of triplicate determinations
(n = 3) ±standard deviation.●, Boiling water； ○, Methanol； ▼, BHA.
78
Table 4.5 Inhibition of lipid peroxidation activity of various extracts from B. javanica.
Specimens
Inhibition of lipid
peroxidation
(%)1,2
IC501,3
(μg/ml)
Boiling water
95.9 ± 0.6 a
354.0 ± 4.8a
Methanol
94.4 ± 1.2 a
187.1 ± 7.2b
Ethyl acetate
14.8 ± 1.2 b
BHA
95.1 ± 0.8 a
ND4
87.3 ± 0.7c
1. Each value was expressed as mean ± standard deviation (n=3). Values within the
column followed by different letter by Duncan’s method were significantly
different (p< 0.05).
2. The concentrations of B. javanica extract and butylated hydroxylanisole (BHA) as
a positive control was 1000 μg/ml.
3. IC50 meant the concentration (μg/ml) of sample required to scavenge 50% of
inhibition of lipid peroxidation.
79
lecithin with Fe2+/ascorbate/H2O2 at pH 7.4 and cause rapid production of
hydroxyl radicals, which abstracted hydrogen atoms from lipid molecule and
resulted in the propagation of lipid peroxidation. Meanwhile, the results
obtained above showed methanol extract exhibited high iron chelating activity.
Methanol extract from B. javanica capable of inhibiting lipid peroxidation might
be due to scavenge the generation of the hydroxyl radicals and chelating metal
ions.
4.1.3 Contents of antioxidant components
Phenolic compounds in plants such as flavonols, tannins and phenolic
acids possessed antioxidant activity to their structure, especially of hydroxyl
groups. The antioxidant activity of B. javanica, such as scavenging free radicals
of DPPH·, ABTS·+ and superoxide anion, reducing Fe3+, chelating Fe2+ and
inhibition of lipid peroxidation was probably resulted from the phenolic
compounds it contained. Plants are a very significant part and represent an
important source of bioavailable polyphenolic compounds such as flavonols,
tannins and phenolic acids, etc (Hatano et al., 1989). The amounts of total
phenolics, flavonids and condensed tannins of B. javanica extracts were
determined and are presented in Table 4.6.
The variation of total phenolics, flavonids and condensed tannins in the
extracts was significant. The total phenolic contents of B. javanica extracts
ranged from 231.5 to 429.6 mg GAE /g. The highest was found in methanol
extract, indicating that the methanol was a more effective way of extracting
phenolic compounds from B. javanica than boiling water and ethyl acetate. In
contrast, the contents of flavonoids in B. javanica extracts were relatively few
80
Table 4.6 Contents of total phenolics, flavonoids, and condensed tannins in various extracts from B. javanica.
Extraction solvent
Total phenolic content 1,2
(GAE mg/ g)
Flavonoids1,2
(QE mg/ g)
b
13.0 ± 0.1
Condensed tannins 1,2
(CE mg/ g)
b
170.8 ±
5.34 b
Boiling water
360.7 ± 19.8
Methanol
429.6 ± 15.7 a
9.6 ± 0.1 c
213.5 ±
9.84 a
Ethyl acetate
231.5 ± 5.7 c
15.4 ± 0.2 a
63.7 ±
9.56 c
1. Values within the column followed by different letter (by Duncan’s method) were significantly different (p< 0.05).
2. GAE: Gallic acid equivalent; QE: Quecetin equivalent; CE: Catechin equivalent.
81
ranging from 9.62 to 15.43 QE mg /g, and the highest was found in ethyl acetate,
followed by boiling water and methanol. Ethyl acetate was seemed to be more
suitable for extracting flavonoids from B. javanica. It was noteworthy that the
amounts of condensed tannin were ca. half of the content, in boiling water and
methanol extracts, 170.8 and 213.5 CE mg/g, respectively. Methanol extract of
B. javanica, was the richest in condensed tannin.
4.1.4 Antioxidant activity of different solvents of B. javanica
The antioxidant activities and contents of ingredients of B. javanica were
summarized in Table 4.7. According to the results above, methanol extract of B.
javanica had the strongest TEACs of DPPH, ABTS, superoxide radicals
scavenging activity, reducing activity, ferrous ion-chelating activity and
inhibition of lipid peroxidation activity. Meanwhile, it contained the highest
amount of total phenolic and condensed tannins. Therefore, methanol extract of
B. javanica was further fractionated for examining their antioxidant activity of
subfractions.
4.1.5 Antioxidant activity of subfractions from methanol extract of B. javanica
4.1.5.1 Yield of subfraction derived from B. javanica methanol extract
Crude methanol extract of B. javanica was sequentially extracted with
n-hexane, diethyl ether, ethyl acetate, 1-butanol and water and the yields were
shown Table 4.8. Water fraction had the highest yield, followed by 1-butanol
fraction.
DPPH· scavenging activities of crude extract and its subfractions of B.
82
Table 4.7 Antioxidant activity and ingredients contents of various solvent extracts from B. javanica.
Antioxidant capacity1
Contents of ingredients
Specimens
Inhibition of lipid
Radical-scavenging activity
Total phenolics
(GAE mg/ g)
Flavonoids
(QE mg/ g )
FRAP
Condensed tannins
(CE mg/ g )
peroxidation
DPPH·
ABTS·+
O2·-
(μmol TE / g ) 3
(μmol TE /g) 3
(μmol TE /g) 3
Boiling water
360.7
±
19.8 b
13.0
±
0.1 b
170.8
±
5.34 b
4173.6 ± 53.0c
4277.4 ± 31.8c
20636.6 ±
Methanol
429.6
±
15.7 a
9.6
±
0.1 c
213.5
±
9.84 a
5664.9 ± 41.1b
5570.7 ± 253.5b
25931.5 ± 2834.3 a
Ethyl acetate
231.5
±
5.7 c
15.4
±
0.2 a
63.7
±
9.56 c
768.3 ± 23.7d
1224.1 ± 68.2d
Ascorbic acid
ND6
ND6
ND6
5715.1 ± 78.8b
9120.7 ± 423.2a
BHA
ND6
ND6
ND6
7090.0 ± 62.2a
8979.2 ± 99.9a
787.8 b
1022.8 ±
1725.6
±
ND6
Metal chelating
164.9 c
193.8c
(μmol TE / g) 3
3. TE meant trolox equivalent antioxidant activity
4. EDTAE meant EDTA equivalents
5. IC50 meant the concentration (μg/ml) of sample required to scavenge 50% of inhibition of lipid peroxidation.
6. ND meant not available because of insufficient yield to accurately determine antioxidant activity.
83
IC505 (μg /ml)
54.7 c
23.2 ± 1.7 b
354.0
±
4.8a
2685.2 ± 127.7 b
59.2 ± 3.7 a
187.1
±
7.2b
31.6 d
7.5 ± 2.4 c
ND6
7507.8 ± 126.7a
56.6 ± 3.4 a
ND6
98.1a
51.5 ± 4.4 a
1712.3 ±
756.7 ±
7863.0 ±
1. Values within the column followed by different letter by Duncan’s method were significantly different (p＜ 0.05). Each value was expressed as mean ± S.D. (n=3).
2. GAE meant gallic acid equivalent, QE meant quecetin equivalent, CE meant catechin equivalent.
(μmol EDTAE / g ) 4
87.3
±
0.7c
Table 4.8 Yields of derived fractions from methanol extract of B. javanica.
Yield (%)1,2,3
Fraction
n-Hexane
Diethyl ether
Ethyl acetate
1-Butanol
Water
0.1
0.7
2.2
32.5
66.5
±
±
±
±
±
0.0 d
0.1c
0.1c
3.4 b
7.3 a
2. Yield is defined as weight percentage of fraction (g) per gram of methanol extract
of B. javanica.
84
javanica are presented in Fig. 4.3. The crude extract and its fractions were
capable of scavenging DPPH· in a does-dependent mode. Among the five
fractions, the water and ethyl acetate fractions possessed the highest DPPH
scavenging activity with no significant difference (P>0.05). Similar results of
AEAC and TEAC values of DPPH· scavenging activity were also found.
However, DPPH· scavenging activity of water and ethyl acetate fractions in
terms of EC50, AEAC and TEAC was weaker than that of BHA (P<0.05). It
needed to be pointed out that the DPPH· scavenging activity in these two
fractions with EC50 value of 46.7 and 48.5 μg/ml, respectively, were comparable
to that of ascorbic acid (49.0 μg/ml) (P＞0.05) under the experiment al
conditions (Table. 4.9). Similarly, methanol extract and its derived fractions
exhibited a concentration-dependent scavenging activity against ABTS·+ (Fig.
4.3). Among the methanol extract and its fractions, ethyl acetate fraction was the
most efficient ABTS·+ scavenger than others. It was worthy noted that the
ABTS·+ scavenging activity in ethyl acetate fraction with EC50 value of 219.4
μg/ml was significantly lower than those of ascorbic acid (224.9 μg/ml) and
BHA (228.2 μg/ml) (P<0.05) under the experimental conditions (Table 4.9), and
this suggested that the components within this fraction possessed extremely
strong ABTS·+ scavenging activity.
In the cellular oxidation reactions, superoxide anion radical was normally
formed first, which generated other radicals including the most destructive
radical hydrogen radical (Korycka-Dahl, 1978). The methanol extract and it
fractions were responsible for accentuated dose-response capacity on superoxide
anion radical (Fig. 4.3). It is interesting to note that the, except n-hexane fraction,
methanol and its fractions on superoxide anion radical scavenging activity were
85
80
60
40
.
100
20
0
0
200
400
600
80
60
40
.+
100
20
0
0
200
400
600
Superoxide anion radicals scavenging activity (%)
120
100
80
60
40
20
0
-20
0
200
400
600
Fig. 4.3 Radical scavenging activity of derived fractions from methanol extract of B.
javanica. ●, Methanol extract； ○,n-Hexane fraction； ▼, Diethyl ether
fraction；△, Ethyl acetate fraction； ■, 1-Butanol fraction；□, Water
fraction；◆, Ascorbic acid；◇, BHA.
86
Table 4.9 Radical scavenging activity of derived fractions from methanol extract of B. javanica. 1,2,3,4
DPPH
Specimens
Crude extract
n-Hexane fraction
ABTS
Superoxide
EC50
AEAC
TEAC
EC50
AEAC
TEAC
EC50
AEAC
TEAC
(μg/ml)
(μmol AE / g)
(μmol TE / g)
(μg/ml)
(μmol AE / g)
(μmol TE / g)
(μg/ml)
(μmol AE / g)
(μmol TE / g)
50.4 ± 1.2 c
ND5
5298.1 ±
75.5 ±
57.2b
5664.9 ±
41.1 b
372.9 ± 6.1b
3409.3 ± 143.8b
5570.7 ± 253.5b
7.6e
165.8 ±
5.5 e
ND5
ND5
60031.9 ±
5826.1c
12249.7 ±
815.0e
4074.9 ±
116.7d
443.6 ± 19.4a
2910.4 ±
87.1 e
4691.3 ± 153.5 e
412.9 ± 26.7b
37221.1 ±
2561.2 d
14834.4 ± 1246.0d
5872.6
617.1 b
219.4 ± 29.4f
6156.1 ±
81.9 a
9626.4 ± 217.9 a
136.2 ± 4.9e
116111.6 ±
6773.6 a
47758.3 ± 3295.2a
337.3c
326.9 ± 29.0c
4174.4 ±
290.5 d
6699.9 ± 512.1d
158.4 ± 9.8d
89840.4 ±
7652.3 b
34977.7 ± 3722.7b
289.5 ± 28.0d 4391.1 ±
413.8 c
7081.9 ± 729.6 c
155.4 ± 6.2d
91031.1 ±
5701.5 b
35556.9 ± 2773.7b
6238.3 ±
398.3f
ND5
216.3 ± 16.9 c
ND5
Diethyl ether fraction
73.5 ± 2.6a
3731.5 ± 162.5d
Ethyl acetate fraction
48.5 ± 1.1c
5444.5
1-Butanol fraction
56.6 ± 4.8b
4970.3 ± 469.0 c
5429.5 ±
Water fraction
46.7 ± 1.1c
5637.0 ± 176.5b
5886.2 ± 112.6 b
Ascorbic acid
49.0 ± 0.7c
5368.0 ± 109.7b
5715.1 ±
78.8b
224.9 ± 4.5 e
5285.1 ±
243.4 b
9120.7 ± 423.2 b
2677.4 ± 11.6 a
BHA
41.3 ± 0.7d
6155.6 ± 32.8 a
7090.0 ±
62.2a
228.2 ± 2.0 e
5002.4 ±
157.4 b
8979.2 ± 99.9 b
ND5
203.3b
25931.5 ± 2834.3c
2686.2 ±
1725.6 ± 193.8f
ND5
1. Each value was expressed as mean ± S.D. (n=3). Values within the column followed by different superscript by Duncan’s method were significantly different (p<0.05).
87
396.5e
ND5
significantly higher than that of ascorbic acid, especially of ethyl acetate fraction,
implying that they possessed better superoxide anion radical scavenging activities
than ascorbic acid.
With regard to values of EC50, AEAC and TEAC of superoxide anion radical
scavenging activity, ethyl acetate fraction was the highest, followed by crude
methanol extract, 1-butanol fraction, water fraction and diethyl ether fraction.
From the results aforesaid, the ethyl acetate fraction was determined to be the
effective antiradical agent; with AEAC values were 5444.5, 6156.1 and 116111.6
AE μmol/g for DPPH·, ABTS·+ and superoxide, respectively, which corresponded to
5872.6, 9626.4 and 47758.3 TE μmol/g, respectively (Table 4.9).
4.1.5.3 Ferric reducing antioxidant power (FRAP)
Antioxidants can be explained as reductants, and inactivation of oxidants by
reductants can be described as redox reactions (Guo et al., 2003; Oktay et al., 2003).
The ferric reducing antioxidant power (FRAP) of methanol extract and its fractions
was shown in Table 4.10. Among the fractions, ethyl acetate fraction had the highest
reducing antioxidant power of 7732.4 μmol Fe2+/g, which corresponds to 5175.1
trolox μmol/g. However, The FRAP values of all fractions were significantly lower
than those of ascorbic acid and BHA. The aforesaid results demonstrated that ethyl
acetate fraction possessed the highest antioxidant activity as evaluated by
scavenging activity against DPPH·, ABTS·+ and superoxide anion radicals as well as
reducing power (FRAP).
4.1.5.4 Ferrous ion-chelating capacity
Ferrous ion is well known as an effective pro-oxidant in the food systems or
88
Table 4.10 Reducing activity of derived fractions from methanol extract of B. javanica by
FRAP method.
Specimens
Crude extract
n-Hexane fraction
1-Butanol fraction
Water fraction
Ascorbic acid
BHA
1.
FRAP-value1
(μmol Fe2+ /g)
3988.2 ± 191.5e
67.8 ± 68.7g
3621.6 ± 647.4f
7732.4 ± 1205.3b
7099.4 ± 1087.8c
4495.9 ± 669.6d
11222.1 ± 190.0 a
11754.8 ± 147.2 a
( μmol trolox/ g )
1712.3 ± 54.7e
65.3 ± 39.0g
2434.6 ± 433.0f
5175.1 ± 807.2b
4753.1 ± 726.3c
3017.4 ± 449.1d
7507.8 ± 126.7a
7863.0 ± 98.1a
Each value was expressed as mean ± S.D. (n=3). Values within the column followed
by different letter by Duncan’s method were significantly different (p<0.05).
89
organism. Antioxidants capable of chelating pro-oxidant metal ions, prevents radical
formation from these pro-oxidants (Kulkarni et al., 2007; Hsu et al., 2003). As
shown in Table 4.11, methanol extract of B. javanica showed a better metal
chelating activity than those of all its derived fractions. The metal chelating
activities of all fractions were as follows: 1-butanol fraction > water fraction, diethyl
ether fraction and ethyl acetate fraction > n-hexane fraction. These results indicated
that the fractions of methanol extract in B. javanica showed metal chelating activity
were lower than that of crude extract.
The crude extract and its derived fractions of B. javanica were capable of
preventing the formation of MDA-(TBA) 2 chrmogen in a does-dependent fashion,
signifying that they had excellent inhibition of lipid peroxidation (Fig. 4.4). At a
concentration of 1000 μg/ml, all the fractions, except n-hexane fraction and diethyl
ether fraction, produced ca. 92-95% of inhibition, similar to BHA. The IC50 values
of fractions except n-hexane fraction increased in the following order: 1-butanol
fraction (139.2 μg/ml) > water fraction (248.0 μg/ml) > ethyl acetate fraction (315.3
μg/ml) > diethyl ether fraction (1128.6 μg/ml) (Table 4.12). It was noted that the
IC50 values of 1-butanol fraction was lower than of methanol extract, indicating
component with higher activity of inhibiting lipid peroxidantion were primarily
separated methanol extract of B. javanica. The pronounced inhibition of lipid
peroxidation may be either due to chelating metal ions or quenching radicals to
terminate chain reaction (Halliwell and Chirico, 1993). Punicalagin found that B.
javanica (Tanaka et al. 1995) and pomegranate fruit (Kulkarni et al., 2004) showed
potential DPPH· and superoxide anion radical scavenging activities and lipid
90
Table 4.11 Ferrous ion-chelating capacity of derived fractions from methanol extract of B.
javanica.
Specimens
(μmol EDTAE / g)
Crude extract
n-Hexane fraction
1-Butanol fraction
Water fraction
Ascorbic acid
BHA
59.3 ±
13.6 ±
18.4 ±
17.4 ±
34.6 ±
19.1 ±
56.6 ±
51.5 ±
1.2 a
1.7 d
0.6 c
1.2 c
1.7 b
1.7 c
3.4 a
4.4 a
91
100
80
60
40
20
0
0
200
400
600
800
1000
1200
Fig. 4.4 Inhibition of lipid peroxidation activity of derived fractions from methanol
extract of B. javanica by TBARS method. Values are means of triplicate
determinations (n = 3) ± standard deviation. ●, Methanol extract；○, Diethyl ether
fraction； ▼, Ethyl acetate fraction； △, 1-Butanol fraction； ■, Water fraction；
□, BHA.
92
extract of B. javanica.
Specimens
Inhibition of lipid
peroxidation activity
(%)1,2
IC501,3
(μg/ml)
Crude extract
94.4 ± 1.2 a
n-Hexane fraction
19.0 ± 0.2 c
45.6 ± 3.4 b
1128.6 ± 60.9 a
92.9 ± 0.4 a
315.3 ± 16.6 d
1-Butanol fraction
96.3 ± 1.7 a
139.2 ± 6.8 e
Water fraction
95.3 ± 1.5 a
248.0 ± 2.2 c
BHA
95.1 ± 0.8 a
87.3 ± 0.7 e
187.1 ± 7.2 d
ND4
column followed by different letter by Duncan’s method were significantly different
(p< 0.05).
2. The concentrations of extract of herbal medicines and butylated hydroxylanisole
(BHA) as a positive control were 1000 μg/ml.
3. IC50 meant the concentration (μg/ml) of sample required to inhibition 50% of lipid
peroxidation activity.
93
peroxidation inhibitory activity.
4.1.5.6 Contents of ingredients subfractions of methanol extract form B. javanica
Plant foods rich in phenolic compounds, especially flavonoids and tannins,
have often been associated with decreased risk of developing diseases, including
cancer, heart diseases, hypertension and stroke, etc. (Silva et al., 2002). Flavonoids
and tannins have powerful antioxidant activities in vitro, being able to scavenge a
wide range of free radicals, such as superoxide, hydroxyl radical, peroxyl radicals,
hypochlorous acid, and peroxynitrous acid, as well as decreasing metal ion
prooxidant activity by chelating metal ions (Silva et al., 2002; Youdim et al., 2002).
In this study, total phenolics, flavonoids and condensed tannins of the fractions of B.
javanica methanol extract are shown in Table 4.13. The highest total phenolic
content was found in ethyl acetate fraction, ca. 358.9 GAE mg/g, followed by water
fraction, 1-butanol fraction, diethyl ether fraction, n-hexane fraction. On the other
hand, the diethyl ether fraction and ethyl acetate fraction had higher contents of
flavonoids, while the lowest was observed in the n-hexane fraction. This result
indicated that moderate polar solvent (diethyl ether and ethyl acetate) is a preferred
solvent for extracted flavonoids. A highest content of condensed tannin was
observed in 1-butanol fraction.
In summary, the antioxidant activities and contents of ingredients of
subfractions of methanol extract from B. javania are shown in Table 4.14.
According to the results, ethyl acetate fraction derived from methanol extract of B.
javania had the highest antioxidant activity including DPPH·, ABTS·+, superoxide
radicals scavenging activity and reducing activity as well as highest contents of total
phenolics and flavonoids.
94
Table 4.13 Total phenolics, flavonoids, and condensation tannins of derived fractions from methanol extract of B. javanica.
Specimens
n-Hexane
Diethyl ether
Ethyl acetate
1-Butanol
Water
Total phenolics1,2
Flavonoids1,2
(GAE mg/ g)
(QE mg/ g)
(CE mg/ g)
e
c
8.3 ± 0.6
12.8 ± 0.6 a
12.6 ± 0.5 a
9.0 ± 0.5 b
9.1 ± 0.5 b
70.7 ± 8.0
243.7 ± 10.6 d
358.9 ± 7.9 a
283.9 ± 10.7 c
337.2 ± 15.8 b
40.5
64.9
249.0
295.5
227.3
1. Values within the column followed by different letter by Duncan’s method were significantly different (p< 0.05).
2. GAE: Gallic acid equivalent.; QE: Quecetin equivalent.; CE: Catechin equivalent.
95
±
±
±
±
±
2.8 d
8.1 c
23.8 b
10.9 a
21.8 b
Table 4.14 Antioxidant capacity and ingredients contents of derived fractions from methanol extract of B. javanica.
Specimens
Total phenolics
FRAP
Flavonoids
tannins
(GAE mg/ g)
(QE mg/ g )
DPPH·
ABTS·+
O2·-
(CE mg/ g )
(μmol TE / g ) 3
(μmol TE /g) 3
(μmol TE /g) 3
165.8 ±
70.7
±
8.0 e
8.3 ± 0.6 c
40.5 ± 2.8 d
243.7
±
10.6 d
12.8 ± 0.6 a
64.9 ± 8.1 c
4074.9 ± 116.7d
4691.3 ± 153.5e
358.9
±
7.9 a
12.6 ± 0.5 a
249.0 ± 23.8 b
5872.6 ± 617.1 b
9626.4 ± 217.9a
1-Butanol fraction
283.9
±
10.7 c
9.0 ± 0.5 b
295.5 ± 10.9 a
5429.5 ± 337.3c
Water fraction
337.2
±
15.8 b
9.1 ± 0.5 b
227.3 ± 21.8 b
n-Hexane fraction
Inhibition of
Condensed
5.5 e
ND5
Metal chelating
lipid
peroxidation
(μmol TE / g) 3
IC505 (μg /ml)
396.5d
65.3 ±
39.0f
13.6 ± 1.7 d
ND6
14834.4 ± 1246.0c
2434.6 ±
433.0e
18.4 ± 0.6c
1128.6 ± 60.9 a
47758.3 ±
3295.2a
5175.1 ±
807.2b
17.4 ± 1.2 c
315.3 ± 16.6 c
6699.9 ± 512.1d
34977.7 ±
3722.7b
4753.1 ±
726.3c
34.6 ± 1.7b
139.2 ±
6.8 d
5908.3 ± 126.7 b
7081.9 ± 729.6 c
35556.9 ±
2773.7b
3017.4 ±
449.1d
19.1 ± 1.7 c
248.0 ±
2.2 b
1725.6 ±
193.8e
7507.8 ±
126.7a
56.6 ± 3.4 a
98.1a
51.5 ± 4.4 a
Ascorbic acid
ND6
ND6
ND6
5715.1 ±
78.8b
9120.7 ± 423.2 b
BHA
ND6
ND6
ND6
7090.0 ±
62.2a
8979.2 ± 99.9 b
2686.2 ±
ND6
7863.0
±
1. Values within the column followed by different letter by Duncan’s method were significantly different (p＜ 0.05). Each value was expressed
as mean ± S.D. (n=3).
4. EDTAE meant EDTA equivalents.
96
ND6
87.3 ±
0.7 d
The high tested antioxidant activity might be related with the hydroxyl groups
and substitution with electron /hydrogen -donating alkyl or methoxy groups of
phenolic compounds (Robards et al., 1999) in ethyl acetate fraction of B. javania.
Moreover, the 1-butanol fraction had the highest activity in Fe2+-chaletion and
inhibition of lipid peroxidation as well as the highest contents of condensed tannins.
This result suggested that high contents of condensed tannins in 1-butanol fraction
were capable of cheating Fe2+, these displayed high inhibitory activity of lipid
peroxidation (Nakamura et al., 2003).
4.1.5.7 Correlations of antioxidant activities with contents of antioxidant compounds
Significant correlations between antioxidant properties and total phenolic for
plant foods have been previously reported in numerous studies. There are significant
correlations between DPPH· scavenging activity and total phenolics in wheat (Li et
al., 2005; Moore et al., 2006) and in juice of citrus (Xu et al., 2008). In this study,
analyzed the antioxidant activities and each ingredient contents in each of fractions
derived from B. javania extract was correlated by Pearson correlation analysis and
the results are shown in Table 4.15. Highly significant correlation coefficient (0.777)
was obtained between condensed tannins and total phenolic, indicating condensed
tannins was the major construction of phenolic compounds in each fraction. In
comparison with total phenolics contents and tested antioxidant activities, except
chelating activity, in each fraction derived from B. javania extract, high correlation
coefficients ranging from 0.709 to 0.982 were found. Surprisingly, there were no
significant correlations between flavonoids content and tested antioxidant activity.
This indicated that total phenolic was good indicator and flavonoids played a
minimal role in the antioxidant activity for B. javania.
97
Table 4.15 Correlation coefficients between assays in fractions of methanol extract in B. javania.
ABTS radical
Superoxide anion
cation
radical
Inhibition of
DPPH radical
Condensed
Total phenolics
Flavonoids
scavenging
tannins
Metal chelating
lipid
activity
peroxidation
FRAP
scavenging
scavenging
activity
activity
activity
Total phenolics
1.000
Flavonoids
0.423
1.000
Condensed tannins
0.777(**)
0.982(**)
0.959(**)
0.923(**)
0.861(**)
0.323
-0.036
0.363
0.471
0.144
0.369
-0.202
1.000
0.811(**)
0.823(**)
0.928(**)
0.865(**)
0.666(**)
0.955(**)
1.000
0.943(**)
0.951(**)
0.862(**)
0.459
0.762(**)
1.000
0.907(**)
0.890(**)
0.352
0.723(**)
1.000
0.880(**)
0.615(*)
0.903(**)
1.000
0.557(*)
0.723(**)
1.000
0.670(**)
DPPH radical scavenging activity
ABTS radical cation scavenging activity
Superoxide anion radical scavenging activity
FRAP
Metal chelating activity
Inhibition of lipid peroxidation activity.
1.
(**) meant ignificant
activity
0.709(**)
-0.240
1.000
at P < 0.01. (*) meant significant at P < 0.05.
98
Content of condensed tannins correlated highly (p < 0.01) with scavenging
activities against DPPH·, ABTS·+ and superoxide, FRAP, and ferrous
ion-chelating activity and inhibition of lipid peroxidation indicating that
condensed tannins played a major role for the antioxidant capacity of B. javania.
High correlations of DPPH radical scavenging activity and reducing power with
total phenolics were also found in beers and wines (Lugasi and Hóvári, 2003).
Correlations were tested to link the antioxidative activity measured by
different assays with each other. Table 4.15 summarized the Pearson’s
correlation coefficients between all analyses carried out in the fractions of
methanol extract from B. javania. DPPH·, ABTS·+ and superoxide anion radical
scavenging activities, reducing power and inhibition of lipid peroxidation assay
were well positively correlated with each other, but metal chelating activity
exhibited weak correlation with DPPH· and ABTS·+ scavenging activities. This
suggested that the compounds in B. javania which could scavenge DPPH·,
ABTS·+ and superoxide were also to reduce ferric ions and inhibited of lipid
peroxidation. This results was supported by Surveswaran et al. (2007) who
showed highly significant linear correlation between DPPH· and ABTS·+
radicals scavenging activities and ferric ion reducing ability in 133 Indian
medicinal.
4.1.6. Conclusion
B. javania has been used as a medicinal plant in Taiwan. To our best
knowledge, this is the first report demonstrating that the antioxidant activity of B.
javania. The methanol extract was observed to possess strongest antioxidant
effects with high content of condensed tannins. Five fractions, included
99
n-hexane, diethyl ether, ethyl acetate, 1-butanol and water fractions, which
fractionated from methanol extract by liquid-liquid partition. Ethyl acetate
fraction showed excellent activity of DPPH·, ABTS·+ and superoxide anion
radical scavenging activities, and FRAP. However, the highest activity of
chelating Fe2+ and inhibiting of lipid peroxidation was found in 1-butanol
fraction. The correlation coefficient from Pearson correlation analysis indicated
a positive relation between antioxidant activity and content of condensed
tannins.
4.2 Polygonum chinensis Linn
4.2.1 Extraction yields from different solvents
It has been previously reported that extraction solvents with different
polarity significantly affect the antioxidant activity of plants (Julkunen-Tiito,
1985; Marinova and Yanishlieva, 1997). Water, methanol and ethyl acetate are
widely used as solvents for extracting antioxidative components in plants (Tsao
and Deng, 2004). In addition boiling water can extract more polyphenols in
plants and herbs (Lim and Murtijaya, 2007). In this study, water, methanol and
ethyl acetate with different polarity were used for extracting antioxidants in P.
chinensis. As shown in Table 4.16, the yields of extracts with different solvents.
The significantly highest yield was obtained in methanol extract, followed by
boiling water and ethyl acetate. Similar phenomenon was reported by
Jayaprakasha et al. (2007), indicating that yields of extraction with different
polarity solvents of Cinnamomum zeylanicum fruits were in a descending order
of methanol, water, ethyl acetate and acetone. This might be caused by the
differences in polarity of extraction solvents (Tsao and Deng, 2004).
100
Table 4.16 Extraction yield of P. chinensis extracts from different solvents.
Extraction yield (%)1,2,3
Solvents
Boiling water
19.7 ± 0.6 b
Methanol
23.3 ± 2.1 a
1.1 ± 0.4 c
Ethyl acetate
2. Extraction yield is defined as weight percentage of extract (g) per gram of dry
weight of P. chinense.
101
4.2.2. Antioxidant activities of various extracts from P. chinenise
Radicals has been implicated in various pathological conditions involving
neurodegenerative disorders, cancer, liver cirrhosis, cardiovascular diseases,
atherosclerosis, cataracts, diabetes, inflammation and ageing (Sayre et al., 2001).
Scavengers of free radicals play an essential role in protection of oxidative
damage (Javanmardi et al., 2003; Kirakosyan et al., 2003). Most widely used
synthetic radicals for evaluating scavenging activity of antioxidants were DPPH·,
ABTS·+ and superoxide anion (O2·-) radicals due to their stability at room
temperature and operational simplicity (Huang et al., 2005). In addition,
scavenging activity of antioxidants differs in the radical system used for
evaluation, and two and more radical systems are required to estimate the
scavenging activity of test samples (Wang et al., 1998; Yu et al., 2002).
Therefore, we examined the scavenging activities of P. chinensis extracts from
different solvents against DPPH·, ABTS·+ and O2·-.
Fig. 4.5 showed the DPPH· scavenging activities of P. chinensis extracted
with different solvents. The DPPH· scavenging activity of all the test extracts
increased with increasing of the extracts, indicating a does-dependent
scavenging mode. Among them, methanol extract exhibited the strongest of
DPPH·, followed by boiling water and ethyl acetate ones, and the scavenging
activities were 81.9, 61.0, and 11.9%, respectively, at a concentration of 100
μg/ml. It is worthy of note that the scavenging activities of methanol extract
against DPPH· was similar to that of ascorbic acid.
Chen et al. (1999) reported that P. multiflorum, belonging to the same
102
80
60
40
.
100
20
0
0
200
400
600
800
1000
.+
100
80
60
40
20
0
0
1000
2000
3000
4000
100
80
60
40
20
0
-20
0
200
400
600
800
1000
Fig. 4.5 Radical scavenging activities of different extracts of P. chinensis.●, Boiling water； ○,
Methanol； ▼, Ethyl acetate；△, Ascorbic acid；■, BHA.
103
genus of P. chinensis, showed stronger scavenging activity against DPPH· than
those of gallic acid and catechin. Similar inhibition effects of P. chinense
extracts against ABTS·+ scavenging activity (Fig. 4.5). Methanol extract
possessed the strongest activity. However, the ability of reducing ABTS·+ of P.
chinensis was not as effective as that of quenching DPPH· radicals. At the
concentration of 600 μg/ml, the scavenging activities of methanol, boiling water
and ethyl acetate extracts were 76.5, 45.3, and 8.6%, respectively. With regard
to superoxide anion radicals (O2·-) of P. chinensis extracts (Fig. 4.5), A
does-dependent inhibition was also found. Methanol and boiling water extracts
exhibited similar activities, 96.4 and 94.0 %, respectively, at a concentration of
1000 μg/ml. It was clearly observed that their activity were significantly higher
than that of ascorbic acid. Similar phenomenon was reported by Qi et al. (2006)
indicating polysaccharide extracted from Ulva pertusa and ascorbic acid, the
scavenging activity against O2·- was about 90％ and 40％, respectively, at the
concentration of 800 μg/ml.
In order to further evaluate the scavenging activities against DPPH·,
ABTS·+ and O2·- of different extracts from P. chinensis, EC50 (concentration for
reducing 50% of radicals), ascorbic acid equivalent antioxidant activity (AEAC)
and trolox equivalent antioxidant activity (TEAC) were determined. A lower
EC50 value or higher AEAC and TEAC value is associated with high radical
scavenging activity. As indicated in Table 4.17, methanol extract had the highest
radicals scavenging activity whereas ethyl acetate extract the lowest. The EC50
value of DPPH· scavenging activity was about 54.3 μg/ml, which corresponds to
232.8 μg of P. chinensis and was not significantly different to that of ascorbic
104
Table 4.17 Radical scavenging activity of various extracts from P. chinensis extracts.1,2,3,4
Specimens
DPPH
EC50
(μg/ml)
AEAC
TEAC
(μmol AE/g) (μmol TE/g)
O2·-
ABTS
EC50
(μg/ml)
AEAC
(μmol AE/g)
TEAC
(μmol TE/g)
EC50
(μg/ml)
AEAC
(μmol AE/g)
TEAC
(μmol TE/g)
734.6 ± 8.9b
1610.3 ± 40.0 c
2575.0 ± 70.5 c
252.8 ± 24.3c
52514.2 ± 3420.0b
22274.3 ± 1663.8 b
Boiling water 75.6 ± 4.2 b 3600.0 ± 112.3 c 3999.0 ± 49.1c
54.3 ± 0.8 c
5218.4 ± 450.7b 5607.7 ± 323.6b
400.3 ± 37.6c 3077.9 ± 59.3 b 4986.6 ± 104.6 b
232.5 ± 7.9 b
56117.2 ± 2555.8a
24027.1 ± 1243.3 a
Methanol
ND5
5134.4 ± 1346.0d
1188.5 ± 654.8 d
Ethyl acetate 462.6 ± 8.4 a 662.0 ± 59.9 d 684.3 ± 32.7d 3721.8 ± 85.2a 485.4 ± 28.7d 592.1 ± 50.6 d
224.9 ± 4.5 d
5285.1 ± 243.4 a 9120.7 ± 423.2 a
2677.4 ± 11.6 a
6238.3 ±
398.3c
1725.6 ± 193.8c
Ascorbic acid 49.0 ± 0.7 c 5368.0 ± 109.7b 5715.1 ± 78.8b
5
ND
41.3 ± 0.7d
6155.6 ± 32.8 a
7090.0 ± 62.2a
228.2 ± 2.0 d
5002.4 ± 157.4 a 8979.2 ± 99.9 a
ND5
ND5
BHA
1. Each value was expressed as mean ± S.D. (n=3). Values within the column followed by different superscript by Duncan’s method were
significantly different (p<0.05).
105
acid (P >0.05), naming the methanol extract of P. chinensis has similar DPPH·
scavenging activity to ascorbic acid. AEAC of P. chinensis extracts ranged from
662.0 to 5218.4 μmol AE / g, while TEAC ranging from 684.3 to 5607.7 μmol
TE / g.
Antioxidants can be explained as reductants, capable of donating electrons.
The ferric reducing antioxidant power assay (FRAP) can determine the ability of
the antioxidants in reducing Fe3+ to Fe2+ (Huang et al., 2005). Table 4.18
shows the reducing power of P. chinensis extracts from different solvent and
expressed as μmol Fe2+ and trolox equivalent /g extract. Reducing activity of all
extracts was found but with significant difference. Methanol extract of P.
chinense showed the highest FRAP-values of 4031.5 μmol Fe2+ /g extract and
2714.1 μmol trolox /g extract, indicating that methanol extract of P. chinensis
had the stronger reducing activity than the other extracts. However, the
FRAP-value of methanol extract were much lower than those of ascorbic acid
(11222.1 μmol Fe2+ /g and 7507.8 μmol trolox /g) and BHA (11754.8 μmol Fe2+
/g and 7863.0 μmol trolox /g) under the same experimental conditions.
It has been well recognized that chelating agents with ability of quenching
transition metal ions inhibits the formation of the first few free radicals and
consequently reduce radical damage and lipid peroxidation (Korycka-Dahl,
1978; Zhao et al., 2008). To better evaluate the potential antioxidant properties
of P. chinensis chelating activity of various extracts, against Fe2+ was examined
and expressed as EDTA equivalents per gram of extract. As shown in Table 4.19,
all P. chinensis extracts exhibited ferrous ion-chelating activity at test
concentration, but significantly differenced in their activities methanol extract
with the highest ferrous ion-chelating activity of 55.9 μmol EDTA equivalents/g,
followed by ethyl acetate and boiling water extract of 22.1 and 1.7 μmol EDTA
equivalents /g, respectively, under the experimental conditions, the results
indicated that methanol is a preferred solvent for P. chinensis of
ferrous
ion-chelating agents. As comparison, Huang et al. (2008) found that methanol
106
Table 4.18 Reducing activity of P. chinensis extracts using FRAP method.
FRAP-value1,2
Specimens
(μmol trolox / g)
(μmol Fe2+ /g)
Boiling water
1925.9 ± 80.7 c
1310.4 ± 53.8c
Methanol
4031.5 ±177.0 b
2714.1 ± 118.0b
Ethyl acetate
229.3 ± 12.5 d
179.3 ±
8.3d
Ascorbic acid
11222.1 ±190.0 a
7507.8 ± 126.7a
BHA
11754.8 ±147.2 a
7863.0 ± 98.1a
0.05).
107
Table 4.19 Ferrous ion-chelating capacity of various extracts from P. chinensis.
Specimens
(μmol EDTAE / g)
Boiling water
1.7 ± 0.2 b
Methanol
55.9 ± 1.4 a
Ethyl acetate
22.1 ± 0.5 c
Ascorbic acid
56.6 ± 3.4a
BHA
51.5 ± 4.4 a
0.05).
2. EDTAE standard for EDTA equivalents.
108
extracts of four P. species showed much low ferrous ion-chelating activity than
quercitrin.
Lipid peroxidation plays an important role in oxidative stress in biological
systems. Because of several toxic byproducts from the peroxidation can damage
other bio-molecules. Antioxidants acting in living system are classified into
preventive antioxidants and chain-breaking (Liu and Ng, 2000). The inhibitions
of lipid peroxidation of various P. chinensis extracts assayed by the TBARS test
are presented in Fig 4.6.
The poorest inhibition was found in ethyl acetate. The results indicated
that P. chinensis extracts dissolved in polar solvents were capable of protecting
lecithin from oxidative degradation by scavenging hydroxyl radicals. However,
the ability of anti-lipid peroxidation of methanol and boiling water extracts with
IC50 values (concentration for inhibition 50% of lipid peroxidation) of 250.5
μg/ml and 324.3 μg/ml, respectively, was not comparable to that of BHA (87.3
μg/ml) (Table 4.20).
4.2.3 Contents of antioxidant components
The results obtained above demonstrated that P. chinensis extracts with
antioxidant activity could quench free radicals of DPPH·, ABTS· + and
superoxide anion, reducing Fe3+ and chelate Fe2+. These antioxidant activities
might be resulted from the phenolic compounds in P. chinensis. Phenolic
compounds contained large varieties such as flavonoids and condensed tannins
(Cai et al., 2006). To better understand the relationship between the antioxidant
activity and phenolic compounds in P. chinensis, the amounts of total phenolics,
flavonids and condensed tannin of the P. chinensis extracts were determined and
are presented in Table 4.21. Significant amounts of total phenolics were detected
in all P. chinensis extracts, ranging from 259.5 to 540.7 mg GAE /g. This might
be due to the differences in polarity of the extraction solvents. A highest content
of total phenolics was observed in methanol extract of P. chinensis followed by
boiling water and ethyl acetate extracts. However, the contents of flavonoids in
109
Inhibition of lipid peroxidation (%)
100
80
60
40
20
0
0
200
400
600
800
1000
Fig. 4.6 Inhibition of lipid peroxidation of boiling water and methanol extracts from P.
chinensis and BHA. Values are means of triplicate determinations (n = 3)
±standard deviation.●, Boiling water； ○, Methanol； ▼, BHA.
110
Table 4.20 Inhibition of lipid peroxidation activity of various extracts from P.
chinensis.
Inhibition of lipid
IC501,3
Specimens
peroxidation
(μg/ml)
(%)1,2
Boiling water
96.0 ± 1.6 a
324.3 ± 1.8a
Methanol
95.0 ± 1.0 a
250.5 ± 5.5b
Ethyl acetate
BHA
8.3 ± 1.5 b
95.1 ± 0.8 a
ND4
87.3 ± 0.7c
column followed by different letter by Duncan’s method were significantly
different (p< 0.05).
2. The concentrations of P. chinensis extract and butylated hydroxylanisole (BHA) as
a positive control was 1000 μg/ml.
3. IC50 meant the concentration (μg/ml) of sample required to scavenge 50% of
inhibition of lipid peroxidation.
111
Table 4.21 Contents of total phenolics, flavonoids, and condensed tannin in various extracts from P. chinensis.
Extraction solvent
Total phenolic content 1,2
(GAE mg/ g)
Flavonoids1,2
(QE mg/ g)
Condensed tannin 1,2
(CE mg/ g)
Boiling water
373.0 ± 14.0 b
8.5
±
0.1 b
213.5 ± 32.9 b
Methanol
540.7 ± 16.0 a
8.4
±
0.1 b
330.7 ± 17.6 a
Ethyl acetate
259.5 ± 7.3 c
14.1
±
0.4 a
66.7 ± 6.3 c
1. Values within the column followed by different letter (by Duncan’s method) were significantly different (p< 0.05).
2. GAE: Gallic acid equivalent; QE: Quecetin equivalent; CE: Catechin equivalent.
112
P. chinensis extracts were in a small portion of total phenolics, ranging from 8.4
to 14.1 QE mg/g.
It is noteworthy that the amounts of condensed tannin were in boiling
water and methanol extract ca. half or above portion in the total phenolics (Table
4.21). Natural tannins in plant are commonly divided into condensed tannins and
hydrolyzable tannins. Condensed tannins are mainly the oligomers and polymers
(e.g., monomers, dimers, and trimers) of flavan-3-ols (catechin derivatives), also
known as proanthocyanidins. Condensed tannins and hydrolyzable tannins are
powerful antioxidant agents because they possess a great number of hydroxyl
groups, especially any ortho-dihydroxy or galloyl groups (Cai et al., 2006). The
antioxidant capacity of these two extracts, including radical scavenging activity,
reducing power and inhibition of lipid peroxidation, might be contributed to
condensed tannins present in P. chinensis.
4.2.4 Antioxidant activity of different solvents of P. chinensis
The antioxidant activities and antioxidant compounds content of P.
chinensis were summaried in Table 4.22. According to the results from
methanol extract of P. chinensis had the highest TEAC of DPPH, ABTS,
superoxide radicals scavenging activity, reducing activity, ferrous ion-chelating
capacity and inhibition of lipid peroxidation activity. Therefore, methanol
extract of P. chinensis was selected for further investigation.
4.2.5 Antioxidant activity of subfraction from methanol extract of P. chinensis
4.2.5.1 Yield of subfraction derived from P. chinensis methanol extract
Yield of subfraction of crude methanol extracts from P. chinense with
n-hexane, diethyl ether, ethyl acetate, 1-butanol and water was shown in Table
4.23. The yields of 1-butanol and water fractions were significantly higher than
that of n-hexane, diethyl ether or ethyl acetate.
DPPH· scavenging activities of crude extract and its derived fractions of P.
chinensis are presented in Fig. 4.7. The test samples with exception of
113
Table 4.22 Antioxidant activity and ingredients contents of various solvent extracts from P. chinensis.
Specimens
Total phenolics
(GAE mg/ g)
Flavonoids
(QE mg/ g )
Condensed tannins
(CE mg/ g )
DPPH·
ABTS·+
O2·-
(μmol TE / g ) 3
(μmol TE /g) 3
(μmol TE /g) 3
2575.0 ± 70.5c
Boiling water
373.0
±
14.0 b
8.5
±
0.1 b
213.5
±
32.9 b
3999.0 ± 49.1c
Methanol
540.7
±
16.0 a
8.4
±
0.1 b
330.7
±
17.6 a
5607.7 ± 323.6b 4986.6 ± 104.6b
Ethyl acetate
259.5
±
7.3 c
14.1
±
0.4 a
66.7
±
6.3 c
Ascorbic acid
ND6
BHA
ND6
ND6
ND6
1663.8 b
22274.3 ±
24027.1 ± 1243.3 a
684.3 ± 32.7d
592.1 ± 50.6 d
1188.5 ±
654.8 d
ND6
5715.1 ± 78.8b
9120.7 ± 423.2 a
1725.6 ±
193.8c
ND6
7090.0 ± 62.2a
8979.2 ± 99.9 a
ND6
FRAP
Metal chelating
Inhibition of lipid
peroxidation
(μmol TE / g) 3
IC505 (μg /ml)
53.8c
1.7 ± 0.2 b
324.3 ± 1.8a
2714.1 ± 118.0b
55.9 ± 1.4 a
250.5 ± 5.5b
8.3d
22.1 ± 0.5 c
7507.8 ± 126.7a
56.6 ± 3.4 a
98.1a
51.5 ± 4.4 a
1310.4 ±
179.3 ±
7863.0 ±
ND6
ND6
87.3
± 0.7c
as mean ± S.D. (n=3).
6. ND meant not available because of insufficient yield to accurately determine antioxidant activity.
114
Table 4.23 Yields of derived fractions from methanol extract of P. chinensis.
Yield (%)1,2,3
Fraction
n-Hexane
Diethyl ether
Ethyl acetate
1-Butanol
Water
0.4
4.0
4.0
35.8
50.6
±
±
±
±
±
0.4 e
0.3 d
0.4 d
8.4 b
2.2 a
2. Yield is defined as weight percentage of fraction (g) per gram of methanol extract
of P. chinense.
115
DPPH . scavenging activity (%)
100
80
60
40
20
0
0
200
400
600
ABTS
.+
scavenging activity (%)
100
80
60
40
20
0
0
200
400
600
100
80
60
40
20
0
-20
0
200
400
600
Fig. 4.7 Radical scavenging activity of derived fractions from methanol extract of P.
chinensis. ●, Methanol extract； ○,n-Hexane fraction； ▼, Diethyl ether
fraction； △, Ethyl acetate fraction； ■, 1-Butanol fraction；□, Water
fraction；◆, Ascorbic acid；◇, BHA.
116
n-hexane fraction exhibited does-dependent scavenging activities. The
magnitudes were in the following order: ethyl acetate fraction, crude extract >
1-butanol fraction > water fraction > diethyl ether fraction. It needs to be
pointed out that the DPPH· scavenging activity in ethyl acetate fraction with
EC50 value of 44.6 μg/ml was comparable to those of BHA (41.3 μg/ml) and
ascorbic acid (49.0 μg/ml) (P＞0.05) under the experimental conditions (Table.
4.24). The n-hexane fraction had about 22.0% of scavenging activity at a
concentration of 600 μg/ml, which suggested that the compounds in n-hexane
fraction possessed weak DPPH· scavenging activity. There results clearly
indicated that the ethyl acetate fraction exhibit significant DPPH· scavenging
activity among all other fractions.
It is well known that antioxidant with ABTS·+ scavenging activity is due to
their hydrogen donating activity. Scavenging activities against ABTS·+ of P.
chinensis extract and its fractions were examined and shown in Fig. 4.7 (B). All
the test samples, except n-hexane fraction, exhibited significant ABTS·+
scavenging activities with dose-dependent fashions. Ethyl acetate fraction was
found to be the highest, whereas hexane fraction was the least active. At the
concentration of 400 μg/ml, the ABTS·+ scavenging activity decreased in the
following order: ethyl acetate fraction (75.1%), crude extract (46.9%) >
1-butanol fraction (42.2%) > water fraction (29.1%) > diethyl ether fraction
(28.2%), with EC50 values of 274.7, 400.3, 479.0, 675.3 and 698.9 μg/ml,
respectively. The EC50 value of n-hexane fraction was not determined due to its
low yield of fractionation. However, ABTS·+ scavenging activity in crude
extract and its derived fractions were lower than those of BHA and ascorbic
acid.
Superoxide anion (O2·-), one of the reactive oxygen species (ROS), is
generated in the imbalanced redox reaction of human body and has been
implicated in the occurrence of many human diseases, including atherosclerosis,
hypertension, diabetes, cancer and aging (Korycka-Dahl,1978). The P. chinensis
methanol extract and its derived fractions were estimated for their superoxide
anion scavenging activity in the PMS/NADH-NBT system and the results are
117
Table 4.24 Radical scavenging activity of derived fractions from methanol extract of P. chinensis. 1,2,3,4
DPPH
Specimens
Crude extract
n-Hexane fraction
ABTS
EC50
(μg/ml)
AEAC
(μmol AE / g)
54.3 ± 0.8 d
5218.4 ± 450.7c
5607.7 ±
323.6c
119.3 ± 31.9g
225.2 ±
22.9g
ND5
43.1f
1651.7 ±
31. 0 f
698.9 ± 27.1a
ND5
202.7 ± 18.1a
44.6 ± 1.9ef
5931.1 ± 165.7ab
1-Butanol fraction
70.3 ± 3.0c
3908.8 ± 185.6d
1522.8 ±
TEAC
(μmol TE / g)
EC50
(μg/ml)
Superoxide
AEAC
(μmol AE / g)
400.3 ± 37.6c 3077.9 ± 59.3 c
ND5
TEAC
(μmol TE / g)
4986.6
EC50
(μg/ml)
± 104.6 c
ND5
AEAC
(μmol AE / g)
232.5 ± 7.9 c
ND5
TEAC
(μmol TE / g)
56117.2 ±
2555.8b
24027.1 ±
1243.3 b
3890.1 ±
351.7e
255.9 ±
171.1e
1668.0 d
12309.3 ±
811.5 d
1974.3 ±
265.8f
2821.5 ± 468.6f
441.2 ±
10.1b
6614.5 ± 902.2 ab
274.7 ± 14.3d 4859.5 ±
303.6b
7687.9 ± 535.1b
61.2 ±
12.1e
4228.8 ±
462.8d
479.0 ± 37.3b 2835.7 ±
198.3d
4413.1 ± 459.8d
319.4 ±
66.0d
44715.7 ±
3564.5c
18480.4 ±
1734.1c
394.0 d
32030.7 ±
226281.7 ± 35383.9 a
93717.1 ± 17213.8 a
Water fraction
165.5 ± 11.5b
1718.7 ±
64.0e
1792.3 ±
45.9 e
675.3 ± 11.8a
2250.5 ±
508.8e
3235.1 ± 391.9e
469.1 ±
3.9b
30331.0 ±
809.8 d
13119.1 ±
Ascorbic acid
49.0 ± 0.7de
5368.0 ± 109.7bc
5715.1 ±
78.08b
224.9 ± 4.5 e
6669.1 ±
240.1a
9120.7 ± 423.2a
2677.4 ±
11.6 a
6238.3 ±
398.3c
1725.6 ±
BHA
41.3 ± 0.7f
6155.6 ± 32.8a
7090.0 ±
62.2a
228.2 ± 2.0 e
6588.9 ±
56.7a
8979.2 ±
99.9a
ND5
ND5
1. Each value was expressed as mean ± S.D. (n=3). Values within the column followed by different superscript by Duncan’s method were significantly different (p<0.05).
118
ND
193.8c
5
shown in Fig. 4.7. The methanol extract of P. chinensis and test fractions, except
n-hexane fraction, had excellent superoxide anion radical scavenging activity in
dose-dependent manners. It is interesting to note that EC50 values of all fractions
were significantly lower than that of ascorbic acid, indicating that they
possessed better superoxide anion radical scavenging activities than ascorbic
acid. Among them, ethyl acetate fraction exhibited the highest scavenging
activity, followed by crude extract, 1-butanol fraction, water fraction, diethyl
ether fraction and n-hexane fraction. These results revealed that the ethyl acetate
fraction exhibited the strongest antioxidant activity, which was identical to the
results in DPPH· and ABTS·+. This result was similar with Kang et al. (2003), in
this case, their found the ethyl acetate fraction of methonlic extract in
Scutellaria baicalensis exhibited the highest scavenging activity of O2·-.
In order to further evaluate the scavenging activities against DPPH·,
ABTS·+ and O2·- of methanol extract and its derived from P. chinensis, ascorbic
acid equivalent antioxidant activity (AEAC) and trolox equivalent antioxidant
activity (TEAC) were determined and the results are presented in Table 4.24.
The AEAC of methanol extract and it derived quench DPPH· scavenging
activity ranged from 119.3 to 5931.1 μmol AE / g, while TEAC ranging from
225.2 to 6614.5 μmol TE /g. Compared with ascorbic acid, the fractions of
methanol extract in P. chinensis, except ethyl acetate fraction, had lower quench
DPPH· scavenging activity. The AEAC of scavenging activities against ABTS·+
from methanol extract of P. chinensis and its derived ranged from 1974.3 to
4859.5 μmol AE / g, while the TEAC ranged 2821.5 to 7687.9 TE / g. However,
the TEAC of ascorbic acid and BHA were 6588.9 and 6669.1 AE /g,
respectively, while AEAC were 8979.2 and 9120.7 TE /g, respectively.
Comparing the aforesaid results showed that the scavenging activities against
ABTS·+ of methanol extract in P. chinensis and its derived fractions are lower
than ascorbic acid and BHA. AEAC of scavenging activities against O2·- from
methanol extract of P. chinensis and its derived ranged from 3890.1 to 226281.7
AE / g, while TEAC ranging from 225.9 to 93717.1 TE / g. Compared to
ascorbic acid, the fractions of methanol extract in P. chinensis, had higher
quench O2·- scavenging activity.
119
Antioxidants can be explained as reductants, and inactivation of oxidants
by reductants can be described as redox reactions (Guo et al., 2003; Oktay et al.,
2003). The ferric reducing antioxidant power (FRAP) of methanol extract and its
fractions was shown in Table 4.25. Among the fractions, ethyl acetate fraction
had the highest reducing antioxidant power of 4935.3 μmol Fe2+/g, which
corresponds to 3306.7 trolox μmol/g. However, The FRAP values of all
fractions were significantly lower than those of ascorbic acid and BHA. The
aforesaid results demonstrated that ethyl acetate fraction possessed the highest
antioxidant activity as evaluated by scavenging activity against DPPH·, ABTS·+
and superoxide anion radicals as well as reducing power (FRAP).
As shown in Table 4.26, all fractions from methanol extracts of P.
chinensis exhibited metal chelating activities at test concentration, but
significantly differenced in their activities. n-Hexane and diethyl ether had the
highest ferrous ion-chelating activity of 30.3 and 31.1 μmol EDTA equivalents/g,
respective, followed by 1-butanol fraction, water aqueous fraction and ethyl
acetate fraction under the experimental conditions, indicated that non-polar
solvent (n-hexane and diethyl ether ) is a preferred solvent of methanol extract
in P. chinensis for ferrous ion-chelating activity.
Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acid
in the cell membranes that generates a number of degradation products. MDA,
one of the products of lipid peroxidation, has been used widely as lipid
peroxidation an index of lipid peroxidation and marker of oxidative stress
(Janero, 1990). Our experiment proved that incubation of lecithin with
Fe2+/ascorbate/H2O2 at pH 7.4 causes rapid peroxidation, detectable by TBA
method are presented in Fig. 4.8. Methanol extract and its drevied showed a
does-dependent inhibition. Ethyl acetate and 1-butanol fractions produced ca.
92-95% of inhibition, significantly analogous to BHA, at concentration of 1000
μg/ml (Table 4.27). In contrast, the lower inhibition was found in n-hexane
120
Table 4.25 Reducing activity of derived fractions from methanol extract of P.
chinensis by FRAP method.
FRAP-value1
Specimens
( μmol trolox/ g )
(μmol Fe2+ /g)
Crude extract
4031.5 ± 177.0c
2714.1 ± 118.0c
n-Hexane fraction
13.4 ± 12.5f
25.5 ± 9.4f
e
1238.4 ± 24.8
842.2 ± 10.7e
3306.7 ± 28.8b
4935.3 ± 48.5b
1-Butanol fraction
4297.8 ± 68.7c
2881.7 ± 42.3c
Water fraction
1512.4 ± 101.6d
1024.8 ± 70.0d
7507.8 ± 126.7a
Ascorbic acid
11222.1 ± 190.0 a
BHA
11754.8 ± 147.2 a
7863.0 ± 98.1a
2. Each value was expressed as mean ± S.D. (n=3). Values within the column
followed by different letter by Duncan’s method were significantly different
(p<0.05).
121
of P. chinensis.
Specimens
(μmol EDTAE / g)
55.9 ± 1.4a
Crude extract
n-Hexane fraction
30.3 ± 0.9b
31.1 ± 0.9b
14.9 ± 0.8d
1-Butanol fraction
24.3 ± 3.4c
Water fraction
13.9 ± 1.5d
56.6 ± 3.4 a
Ascorbic acid
51.5 ± 4.4 a
BHA
122
fraction. Further investigation the IC50 of fractions, except n-hexane fraction,
were decreased in the following order: diethyl ether fraction (1409.8 μg/ml) >
water fraction (1103.4 μg/ml) > 1-butanol fraction (434.2 μg/ml) > ethyl acetate
fraction (241.1 μg/ml).
Inhibitionof lipid peroxidation by ferruginous ion (copper ion) takes place
either through ferryl-perferryl (cupryl-percupryl) complex or through hydroxyl
radical generation (Gutteridge, 1985). There, inhibition lipid peroxidation could
be caused by the absence of ferryl-perferryl (cupryl-percupryl) complex or by
scavenging hydroxyl radical or chelating metal ions. The aforesaid results
suggested that the inhibition lipid peroxidation mechanism of P. chinensis
extracts was not due to iron chelation and is possibly due to chain termination by
radical scavenging activities.
4.2.5.6 Contents of ingredients subfractions of methanol extract form P.
chinensis
Several epidemiological studies provide evidence of the protective effect
of consumption of plants. The preventive role of these foods is due to their
constituent chemicals, especially the polyphenolic flavonoids, tannins, phenolic
acids, and anthraquinones. The total intake of these phytochemicals through the
food chain can reach up to 1 g/day (Pietta, 2000). The total phenolics, flavonoids
and condensed tannins of methanol extract and it derived of P. chinensis are
shown in Table 4.28. The highest total phenolic content of the fraction in
methanol extract of P. chinensis, ca. 329.3 GAE mg/g, was obtained in ethyl
acetate fraction, followed by 1-butanol fraction, diethyl ether fraction, and water
fraction. The highest content of flavonoids in all the fractions was also as found
in ethyl acetate fraction and a lowest content in the water fraction. Condensed
tannins were the phenolic classes in the fraction of methanol extract P. chinensis,
but their variation was analogous to that of flavonoids, except in n-hexane
fraction. Levels of condensed tannins in the fractions of methanol extract P.
chinensis were in the following order: ethyl acetate fraction > diethyl ether
fraction > 1-butanol > water fraction.
123
Inhibition of lipid peroxidation (%)
100
80
60
40
20
0
0
200
400
600
800
1000
1200
1400
1600
Fig. 4.8 Inhibition of lipid peroxidation activity of derived fractions from methanol
extract of P. chinensis by TBARS method. Values are means of triplicate
determinations (n = 3) ± standard deviation. ●, Methanol extract；○, Diethyl
ether fraction； ▼, Ethyl acetate fraction； △, 1-Butanol fraction； ■, Water
fraction；□, BHA.
124
Table 4.27 Inhibition of lipid peroxidation activity of derived fractions from methanol extract of P. chinensis.
Specimens
(%)1,2
95.0 ± 1.0 a
Crude extract
IC501,3
(μg/ml)
250.5 ± 5.5 d
n-Hexane fraction
28.0 ± 1.9 d
ND4
35.6 ± 3.8 c
1409.8 ± 62.2 a
95.0 ± 0.2 a
241.1 ± 17.8 d
1-Butanol fraction
92.8 ± 2.7 a
434.2 ± 5.5 c
Water fraction
41.6 ± 4.1 b
1103.4 ± 36.2 b
BHA
95.1 ± 0.8 a
87.3 ± 0.7 e
1. Each value was expressed as mean ± standard deviation (n=3). Values within the column followed by different letter by Duncan’s method
were significantly different (p< 0.05).
2. The concentrations of extract of herbal medicines and butylated hydroxylanisole (BHA) as a positive control were 1000 μg/ml.
3. IC50 meant the concentration (μg/ml) of sample required to inhibition 50% of lipid peroxidation activity.
125
Table 4.28 Total phenolics, flavonoids, and condensation tannins of derived fractions from methanol extract of P. chinensis.
Specimens
Total phenolics1,2
Flavonoids1,2
(GAE mg/ g)
(QE mg/ g)
(CE mg/ g)
e
c
n-Hexane
39.4 ± 11.8
7.2 ± 0.5
43.6
c
b
Diethyl ether
239.2 ± 9.0
17.2 ± 1.2
252.8
a
a
21.3 ± 0.5
921.7
Ethyl acetate
329.3 ± 3.7
b
c
1-Butanol
268.1 ± 5.7
7.2 ± 0.7
78.3
d
d
Water
170.3 ± 6.3
4.8 ± 0.4
22.8
1. Values within the column followed by different letter by Duncan’s method were significantly different (p< 0.05).
2. GAE: Gallic acid equivalent.; QE: Quecetin equivalent.; CE: Catechin equivalent.
126
±
±
±
±
±
4.0 d
15.7 b
56.8 a
4.2 c
2.7 e
In summary, the antioxidant activities and antioxidant compounds
content of methanol extract and its derived in P. chinensis was shown in
Table 4.29. According to the results from ethyl acetate fraction of methanol
extract in P. chinensis had the highest TEAC of DPPH, ABTS, superoxide
radicals scavenging activity, reducing activity, ferrous ion-chelating
capacity and inhibition of lipid peroxidation activity. The antioxidant
compounds content, such as phenolics, flavonoids and condensed tannin,
were also found the highest in the ethyl acetate fraction.
compounds
It is well known that phenolic compounds are highly effective against
free radicals and antioxidant. Consequently, the antioxidant activities of
plants extracts are often explained by their antioxidant compounds such
total phenolics, flavonoids and condensed tannins contents with highest
correlation (Bahorun et al, 2004). This was also observed in this study
(Table 4.30). In comparison with assayed antioxidant activities and total
phenolics contents in each fraction derived from P. chinensis extract, except,
ferrous ion-chelating capacity, high correlation coefficients ranging from
0.630 to 0.959 were found. However, the highest correlation was found
between the superoxide anion radicals scavenging activity and total
phenolics. Contents of flavonoids gave strong positive correlations with
antioxidant activities, except reducing activity (FRAP) and ferrous
ion-chelating capacity, ranging from 0.627 to 0.903. Moreover, correlations
between antioxidant activities and contents of condensed tannins were
analogous to that of total phenolics contents. Additionally, flavonoids and
condensed tannins connect showed significant positive correlations with
total phenolic contents, respectively (0.644 and 0.885), suggesting they
were the major phenolic compounds in the methanol extract P. chinensis.
127
Table 4.29 Antioxidant capacity and ingredients contents of derived fractions from methanol extract of P. chinensis.
Specimens
Total phenolics
(GAE mg/ g)
n-Hexane fraction
39.4
Condensed
tannins
Flavonoids
(QE mg/ g )
(CE mg/ g )
±
11.8 e
7.2 ± 0.5 c
43.6 ± 4.0 d
c
b
b
239.2
±
9.0
17.2 ± 1.2
252.8 ± 15.7
329.3
±
3.7 a
21.3 ± 0.5 a
921.7 ± 56.8 a
1-Butanol fraction
268.1
±
5.7 b
7.2 ± 0.7 c
78.3 ± 4.2 c
Water fraction
170.3
±
6.3 d
4.8 ± 0.4 d
DPPH·
ABTS·+
O2·-
(μmol TE / g ) 3
(μmol TE /g) 3
(μmol TE /g) 3
225.2 ±
31.0e
1651.7 ±
d
22.9
ND6
255.9 ±
2821.5 ± 468.6
d
6614.5 ± 902.2 ab
7687.9 ± 535.1b
4228.8 ±
462.8d
4413.1 ± 459.8c
22.8 ± 2.7 e
1792.3 ±
45.9 e
12309.3
±
171.1e
811.5
c
FRAP
Metal chelating
(μmol TE / g) 3
25.5 ±
842.2 ±
9.4f
10.7
e
30.3 ±
Inhibition of
lipid
peroxidation
IC505 (μg /ml)
0.9b
31.1 ± 0.9
b
ND6
1409.8 ± 62.2 a
93717.1 ± 17213.8 a
3306.7 ±
28.8b
14.9 ±
0.8d
241.1 ± 17.8 d
18480.4 ±
1734.1b
2881.7 ±
42.3c
24.3 ±
3.4c
434.2 ± 5.5 c
3235.1 ± 391.9d
13119.1 ±
394.0 c
1024.8 ±
70.0d
13.9 ±
1.5d
1103.4 ± 36.2 b
1725.6 ±
193.8d
7507.8 ± 126.7a
56.6 ±
3.4 a
ND6
98.1a
51.5 ±
4.4 a
87.3 ± 0.7 e
Ascorbic acid
ND6
ND6
ND6
5715.1 ±
78.8b
9120.7 ±
78.0a
BHA
ND6
ND6
ND6
7071.3 ±
254.6a
8979.2 ±
23.6a
ND6
7863.0
±
as mean ± standard deviation (n=3).
128
Table 4.30 Correlation coefficients between assays in fractions of methanol extract in P. chinensis.
Total phenolics
Total phenolics
1.000
Flavonoids
Condensed tannins
Flavonoids
Condensed
tannins
Superoxide
anion radical
scavenging
activity
0.680(**)
0.907(**)
0.915(**)
0.959(**)
1.000
0.885(**)
0.820(**)
0.627(*)
0.656(**)
1.000
0.918(**)
0.822(**)
0.793(**)
0.948(**)
1.000
1.000
ABTS radical cation scavenging activity
Superoxide anion radical scavenging activity
FRAP
0.874(**)
Inhibition of
lipid
peroxidation
activity.
0.630(*)
-0.034
0.903(**)
0.642(**)
-0.400
0.634(*)
0.957(**)
0.847(**)
-0.444
0.659(**)
0.973(**)
0.922(**)
0.931(**)
-0.626(*)
-0.547(*)
-0.521(*)
1.000
0.458
0.535(*)
0.268
0.164
0.426
1.000
Metal chelating activity
Inhibition of lipid peroxidation activity.
Metal chelating
activity
-0.410
1.000
FRAP
(**) meant ignificant
ABTS radical
cation
scavenging
activity
0.644(**)
DPPH radical scavenging activity
1.
DPPH radical
scavenging
activity
1.000
at P < 0.01. (*) meant significant at P < 0.05.
129
The aforesaid results suggested that the antioxidant activities in ethyl
acetate fraction from P. chinensis are likely attributed to the flavonoids and
condensed tannins which converted the oxidized intermediate into the stable
form and disconfirms the oxidation chain reaction. Siddhuraju and Becker
(2007) reported good correlations for processed cowpea (Vigna unguiculata
(L.) Walp) seed extracts when scavenging activity of DPPH·, ABTS·+ and
FRAP-values were compared with total phenolic contents. Similarly, high
correlation has also been reported in guava fruit extracts (Jime`nez-Escrig et
al., 2001). The reducing power of phenolics compounds in peanut hulls and
stem bark of Indian laburnum extracts was associated with antioxidant
activity, specifically scavenging of free radicals (Yen and Duh, 1993 and
Siddhuraju et al., 2002). Zhao et al. (2008) reported positively correlations
for the malting barley extract when DPPH· and ABTS·+ radicals scavenging
activities and ferric ion reducing ability, but all of them negative
correlations with metal chelating activity were compared with total
phenolics.
The antioxidant activities in the plant extracts were focused on
phenolic compounds and the antioxidant activity assays used to assess their
antioxidant activities are based on different radicals and mechanisms of
reaction (Zhao et al., 2008). As illustrated in Table 4.30, DPPH·, ABTS·+
and superoxide anion radicals scavenging activities, ferric ion reducing
ability and inhibition lipid peroxidation activity were well positively
correlated with each other, but all the of them obtained poor correlations
with ferrous ion-chelating activity. This result cued that the biocompounds
which could scavenge DPPH· in the methanol extract of P. chinensis were
also able to quench ABTS·+ and superoxide anion radicals, to reduce ferric
ions and inhibition lipid peroxidation. However, not all of these compounds
were the chelators of ferrous ions. Fabulously, metal chelating activity
showed negative correlations with all other antioxidant activity evaluation
indices in the present study, noting that the fractions in methanol extract of
P. chinensis with higher ferrous ion-chelating activity might have lower
scavenging activity against DPPH·, ABTS·+ and superoxide anion radical,
ferric ion reducing ability and inhibition lipid peroxidation activity. Wong et
130
al. (2006) reported the negative correlations between DPPH radical
scavenging activity, ferric ion reducing ability, and cupric ion chelating
activity in 25 plants.
4.2.6. Conclusion
Overall, the present results have found that the antioxidant activities
of P. chinensis and fractions have a good correlation with total phenolics,
flavonoids and condensed tannins. The ethyl acetate fraction from methanol
extract was observed to possess strongest antioxidant activity with highest
content of total phenolics, flavonoids and condensed tannins. From this
perspective, as well as antioxidant, the ethyl acetate fraction from methanol
extract in P. chinensis appears to be a potential candidate for preventing free
radical damage to cell though scavenging free radicals and inhibition lipid
peroxidation process.
131
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CHAPTER 6 APPENDIXES
(A)
100
y = 0.1398x + 11.075
R2 = 0.992
Scavenging activity (%)
80
60
40
20
0
0
100
200
300
400
500
600
Ascorbic acid concentration (μΜ)
(B)
80
70
y = 0.1947x - 10.662
R2 = 0.9904
60
50
40
30
20
10
0
100
200
300
400
Trolox concentration (μΜ)
Fig. 6.1 Standard curve of DDPH ·. (A) Calibration curve of ascorbic acid equivalents.
(B) Calibration curve of trolox equivalents
144
(A)
100
y = 0.0383x - 0.176
R2 = 0.9915
80
60
40
20
0
0
500
1000
1500
2000
(B)
80
y = 0.0253x - 0.3171
R2 = 0.9908
70
60
50
40
30
20
10
0
0
500
1000
1500
2000
2500
3000
Fig. 6.2 Standard curve of ABTS ·+. (A) Calibration curve of ascorbic acid
equivalents. (B) Calibration curve of trolox equivalents
145
(A)
120
y = 0.0036x - 3.7141
R2 = 0.9933
100
80
60
40
20
0
-20
0
5000
10000
15000
20000
25000
30000
(B)
100
y = 0.0074x + 5.9745
R2 = 0.996
80
60
40
20
0
0
2000
4000
6000
8000
10000
12000
Fig. 6.3
Standard curve of superoxide anion radical. (A) Calibration curve of
ascorbic acid equivalents. (B) Calibration curve of trolox equivalents
146
(A)
3.5
y = 1.6979x + 0.0268
R2 = 0.995
3.0
2.5
OD 593
2.0
1.5
1.0
0.5
0.0
0
2000
4000
6000
FeSO4 concentration (μΜ)
(B)
3.0
y = 0.0006x + 0.0905
R2 = 0.998
2.5
OD 593
2.0
1.5
1.0
0.5
0.0
0
1000
2000
3000
4000
5000
Fig. 6.4 Standard curve of FRAP methol. (A) Calibration curve of FeSO4 equivalents.
(B) Calibration curve of trolox equivalents
147
0.7
0.6
OD 562nm
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
60
80
100
120
140
EDTA concentration (μΜ)
Fig. 6.5 Standard curve of EDTA equivalents.
148
160
180
1.0
y = 1.7434x + 0.0121
R2 = 0.9998
OD 750 nm
0.8
0.6
0.4
0.2
0.0
0
100
200
300
400
500
Fig. 6.6 Standard curve of gallic acid equivalents
149
600
1.8
1.6
y = 83.377x + 0.003
2
R = 0.9997
1.4
OD425 nm
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
5
10
15
20
Fig. 6.7 Standard curve of quecetin equivalents
150
25
1.2
y = 1.6979x + 0.0268
R2 = 0.995
1.0
OD510 nm
0.8
0.6
0.4
0.2
0.0
-0.2
0
200
400
600
Fig.6.8 Standard curve of catechin equivalents
151

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